Integrative Analysis of Transcriptomics and Proteomics for Screening Genes and Regulatory Networks Associated with Lambda-Cyhalothrin Resistance in the Plant Bug Lygus pratensis Linnaeus (Hemiptera: Miridae)

Abstract

The prolonged use of pyrethroid insecticides for controlling the plant bug Lygus pratensis has led to upward resistance. This study aims to elucidate the molecular mechanisms and potential regulatory pathways associated with lambda-cyhalothrin resistance in L. pratensis. In this study, we constructed a regulatory network by integrating transcriptome RNA-Seq and proteome iTRAQ sequencing analyses of one lambda-cyhalothrin-susceptible strain and two resistant strains, annotating key gene families associated with detoxification, identifying differentially expressed genes and proteins, screening for transcription factors involved in the regulation of detoxification metabolism, and examining the metabolic pathways involved in resistance. A total of 82,919 unigenes were generated following the assembly of transcriptome data. Of these, 24,859 unigenes received functional annotations, while 1064 differential proteins were functionally annotated, and 1499 transcription factors belonging to 64 distinct transcription factor families were identified. Notably, 66 transcription factors associated with the regulation of detoxification metabolism were classified within the zf-C2H2, Homeobox, THAP, MYB, bHLH, HTH, HMG, and bZIP families. Co-analysis revealed that the CYP6A13 gene was significantly up-regulated at both transcriptional and translational levels. The GO and KEGG enrichment analyses revealed that the co-up-regulated DEGs and DEPs were significantly enriched in pathways related to sphingolipid metabolism, Terpenoid backbone biosynthesis, ABC transporters, RNA transport, and peroxisome function, as well as other signaling pathways involved in detoxification metabolism. Conversely, the co-down-regulated DEGs and DEPs were primarily enriched in pathways associated with Oxidative phosphorylation, Fatty acid biosynthesis, Neuroactive ligand-receptor interactions, and other pathways pertinent to growth and development. The results revealed a series of physiological and biochemical adaptations exhibited by L. pratensis during the detoxification metabolism related to lambda-cyhalothrin resistance. This work provided a theoretical basis for further analysis of the molecular regulation mechanism underlying this resistance.

Keywords: Lygus pratensis; detoxification; lambda-cyhalothrin; proteome; resistance; transcriptome.

Figure 1

Principal component analysis (PCA) of transcriptome data.

Figure 2

The number of significantly up-regulated and down-regulated genes between lambda-cyhalothrin-resistant and -susceptible strains of L. pratensis: (A) differential gene histogram; and (B) differential gene volcano map.

Figure 3

Phylogenetic evolutionary tree of the Cytochrome P450 gene of L. pratensis (red) with other insects (black). Note: The amino acid sequences of Cimex lectularius (Cl), Apolygus lucorum (Al), Halyomorpha halys (Hh), and Nesidiocoris tenuis (Nl) were analyzed using the MEGA-11 adjacency method. Green is a CYP6 family branch, orange is a branch of the CYP4 family, and blue is a branch of the CYP2 family. The two letters before the gene names are the species name. The topology was tested using bootstrap analyses (1000 replicates), and the number on the branch indicates the confidence coefficient.

Figure 4

Phylogenetic evolutionary tree of Glutathione S-transferase genes of L. pratensis (red) with other insects (black). Note: The amino acid sequences of Cimex lectularius (Cl), Halyomorpha halys (Hh), Laodelphax striatellus (Ls), and Subpsaltria yangi (Sy) were analyzed using the MEGA-11 adjacent method. Green is a branch of the GST-Delta (D) family, red is a branch of the GST-Epsilon (E) family, purple is a branch of the GST-Zeta (Z) family, orange is a branch of the GST-Theta (T) family, and blue is a branch of the GST-Sigma (S) family. The two letters before the gene names are the species name. The topology was tested using bootstrap analyses (1000 replicates), and the number on the branch indicates the confidence coefficient.

Figure 5

Phylogenetic evolutionary tree of ABC transporter genes from L. pratensis (red) with other insects (black). Note: The amino acid sequences of Cimex lectularius (Cl), Halyomorpha halys (Hh), Macrosteles quadrilineatus (Mq), and Nesidiocoris tenuis (Nt) were analyzed using the MEGA-11 adjacency method. Red is a branch of the ABC-A family, orange is a branch of the ABC-B family, blue is a branch of the ABC-C family, purple is a branch of the ABC-F family, and green is an ABC-G family branch. The two letters before the gene names are the species name. The topology was tested using bootstrap analyses (1000 replicates), and the number on the branch indicates the confidence coefficient.

Figure 6

Phylogenetic evolutionary tree of Carboxylesterase genes of L. pratensis (red) with the other insects (black). Note: The amino acid sequences of Cimex lectularius (Cl), Halyomorpha halys (Hh), Nesidiocoris tenuis (Nt), Apolygus lucorum (Al), and Lygus lineolaris (Ll) were analyzed using the MEGA-11 adjacency method. Green is a branch of the Acetylcholinesterase family, and red is a branch of the alpha/beta hydrolases family. The two letters before the gene names are the species name. The topology was tested using bootstrap analyses (1000 replicates), and the number on the branch indicates the confidence coefficient.

Figure 7

Analysis of differentially expressed genes associated with detoxification metabolism resistance: (A) Cytochrome P450 gene differential expression heat map; (B) Glutathione S-transferase gene differential expression heat map; and (C) ABC transporter gene differential expression heat map (red indicates up-regulation; blue indicates down-regulation).

Figure 8

Heat map analysis of differentially expressed transcription factors.

Figure 9

Heat map analysis of transcription factors associated with pyrethroid resistance.

Figure 10

Principal component analysis (PCA) of proteome data.

Figure 11

Validation of differentially expressed genes between RT-qPCR and RNA-Seq: (A) S-VS-R6; (B) S-VS-R14 (note: differentially expressed gene); (C) S-VS-R6; and (D) S-VS-R14 (note: differential expression of transcription factors).

Figure 12

Statistical information of differently expressed proteins: (A,B) differential protein column diagrams; and (C) differential protein volcano diagram.

Figure 13

Proteomic differential protein heat map.

Figure 14

Venn diagram of differentially expressed genes and protein numbers. Note: Genes/proteins outside the threshold lines indicate significant differences, while those inside the threshold lines indicate non-significant differences.

Figure 15

The nine-quadrant diagram analysis of transcriptome and proteome association. Note: Genes/proteins outside the threshold lines indicate significant differences, while those inside the threshold lines indicate non-significant differences. Red dots represent DEGs/DEPs, blue dots represent DEPs/NDEGs, green dots represent NDGEs/DEPs, black dots represent NDGEs/NDEPs, and gray dots represent DEGs/DEPs with p-value > 0.5.

Figure 16

DEGs/DEPs GO enrichment classification: (A) co-up-regulated DEGs/DEPs GO enrichment cycle; (B) co-down-regulated DEGs/DEPs GO enrichment cycle; (C) co-up-regulated DEGs/DEPs GO enrichment column; and (D) co-down-regulated DEGs/DEPs GO enrichment column diagram.

Figure 16

DEGs/DEPs GO enrichment classification: (A) co-up-regulated DEGs/DEPs GO enrichment cycle; (B) co-down-regulated DEGs/DEPs GO enrichment cycle; (C) co-up-regulated DEGs/DEPs GO enrichment column; and (D) co-down-regulated DEGs/DEPs GO enrichment column diagram.

Figure 17

DEGs/DEPs KEGG enrichment classification: (A) co-up-regulated DEGs/DEPs KEGG enrichment cycle; (B) co-down-regulated DEGs/DEPs KEGG enrichment cycle; (C) co-up-regulated DEGs/DEPs KEGG enrichment column; and (D) co-down-regulated DEGs/DEPs KEGG enrichment column diagram.

Figure 17

DEGs/DEPs KEGG enrichment classification: (A) co-up-regulated DEGs/DEPs KEGG enrichment cycle; (B) co-down-regulated DEGs/DEPs KEGG enrichment cycle; (C) co-up-regulated DEGs/DEPs KEGG enrichment column; and (D) co-down-regulated DEGs/DEPs KEGG enrichment column diagram.