ATP-Binding Cassette (ABC) Transporter Genes Involved in Pyrethroid Resistance in the Malaria Vector Anopheles sinensis: Genome-Wide Identification, Characteristics, Phylogenetics, and Expression Profile

Abstract

background: The ATP-binding cassette (ABC) transporters family is one of the largest families of membrane proteins existing in all living organisms. Pyrethroid resistance has become the largest unique obstacle for mosquito control worldwide. ABC transporters are thought to be associated with pyrethroid resistance in some agricultural pests, but little information is known for mosquitoes. Herein, we investigated the diversity, location, characteristics, phylogenetics, and evolution of ABC transporter family of genes in the Anopheles sinensis genome, and identified the ABC transporter genes associated with pyrethroid resistance through expression profiles using RNA-seq and qPCR. Results: 61 ABC transporter genes are identified and divided into eight subfamilies (ABCA-H), located on 22 different scaffolds. Phylogenetic and evolution analyses with ABC transporters of A. gambiae, Drosophila melanogaster, and Homo sapiens suggest that the ABCD, ABCG, and ABCH subfamilies are monophyly, and that the ABCC and ABCG subfamilies have experienced a gene duplication event. Both RNA-seq and qPCR analyses show that the AsABCG28 gene is uniquely significantly upregulated gene in all three field pyrethroid-resistant populations (Anhui, Chongqing, and Yunnan provinces) in comparison with a laboratory-susceptible strain from Jiangsu province. The AsABCG28 is significantly upregulated at 12-h and 24-h after deltamethrin exposure in three-day-old female adults. Conclusion: This study provides the information frame for ABC transporter subfamily of genes, and lays an important basis for the better understanding and further research of ABC transporter function in insecticide toxification. The AsABCG28 gene is associated with pyrethroid detoxification, and it functions at later period in the detoxification process for xenobiotics transportation.

Keywords: ATP-Binding Cassette (ABC) transporters; Anopheles sinensis; RNAseq analysis; deltamethrin; gene expression; pyrethroid resistance.

Figure 1

Conserved domains of A. sinensis ABC transporters. Gray bars represent the amino acid sequences of each structure type of ABC transporter genes. The subfamily names of the ABC transporters are marked on the left side of the structure types, and the gene numbers of each structure type are denoted in brackes on the right side of the gray bars. The purple-filled rectangular boxes denote transmembrane domain (TMD) positions on the amino acid sequences, and the blue-filled oval boxes indicate nucleotide binding domain (NBD) positions. The TMD and NBD domains of each A. sinensis ABC transporter were identified using the program Pfam.

Figure 2

Distribution of A. sinensis ABC transporter genes on scaffolds. The number of left side on scaffold represents physical locations, and on the right side are gene names.

Figure 3

Phylogenetic relationships of the A. sinensis ABC transporter genes. The relationships were inferred based on the amino acid sequences of nucleotide binding domains (NBD) with maximum likelihood method using MEGA5.0, and those genes with two NBDs were treated as two separate gene taxa with each taxon using one different NBD amino acid sequence. The bootstrap values calculated from 1000 replicates were marked on each corresponding node. ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG, and ABCH represent eight different subfamilies in the ABC transporter family.

Figure 4

Expression profile of ABC transporter genes in three pyrethroid-resistent field populations (AH-FR, CQ-FR, and YN-FR) compared with a laboratory-susceptible strain (WX-LS), detected by RNA-seq. Genes with FPKM value ≥ 2 (i.e., ≥ 1 in Log₂, showing on X-axis) and p-value < 0.05 (i.e., > 1.30 in –Log₁₀, showing on Y-axis) are considered to be significantly upregulated, whereas those with FPKM value ≤ 0.5 (≤ −1 in Log₂) and p-value < 0.05 are regarded as downregulated. Vertical dotted lines mark the 1 and −1 of Log₂ (fold changes of FPKM value) values on the X-axis, and horizontal dotted lines denote the p-value = 0.05 of –Log₁₀ (p-value) on the Y-axis. There are six genes that are significantly differently expressed in different populations, in which four genes are upregulated (AsABCG28 commonly in three populations) and three genes are downregulated (AsABCA5 is upregulated in AH-FR but downregulated in CQ-FR). The red-, green-, and blue-filled circles indicate the AH-FR, CQ-FR, and YN-FR, respectively.

Figure 5

qPCR verification of six genes significantly differentially expressed in RNA-seq analysis. (A) The relative expression levels of these genes are shown as the mean ± SD of three biological replicates in qPCR analysis. The S7 (RPS7) and L49 (RPL49) genes were used as internal reference for expression normalization. The population/strain pairs marked with same letters are not significantly different in expression (p-value ≥ 0.05), and the remaining pairs are significantly different (p-value < 0.05), determined by one-way ANOVA. (B) Comparison of expression fold-changes between RNA-seq and qPCR analyses for the six genes in three field populations comparative with the laboratory-susceptible strain WX-LS. AH: Anhui population; CQ: Chongqing; YN: Yunnan.

Figure 6

Expression changes of ten ABC transporter genes after pyrethroid treatment for A. sinensis female adults. The ten genes include four genes (AsABCG28, AsABCA5, AsABCC9, and AsABCG7) that are significantly upregulated in RNA-seq and qPCR analyses in different resistant population, and six genes (AsABCB1, AsABCB2, AsABCB3, AsABCB4, AsABCB5, and AsABCC2) that have been reported to be associated with pyrethroid resistance. The expression levels (means ± SD) were determined at 6-h, 12-h, and 24-h postpyrethroid treatment using qPCR with three biological replicates. The samples marked with “*” were significantly differently expressed (p-value < 0.05) compared with the untreated samples at the same time, and the samples without “*” were not significantly differently expressed.