De Novo Transcriptome Sequencing and Profiling of Ovarian Development of Argas persicus Along the Trophogonic Cycle

Abstract

Background: Argas persicus is a hematophagous ectoparasite of poultry and is the vector of several agents infectious to poultry. This study aims to explore the key genes affecting the ovarian development of A. persicus. Methods: RNA-seq was performed on the ovaries of A. persicus before blood-feeding, on the day of engorgement, and 6 days post-engorgement. Utilizing the threshold padj < 0.05 and|log₂(foldchange)| > 1, differentially expressed genes were identified, and hub genes were determined by constructing protein-protein interaction (PPI) networks. Results: A total of 1008 differentially expressed genes were obtained during the feeding period, including 448 up-regulated and 560 down-regulated genes. Further, 2179 differentially expressed genes were screened in the preoviposition stage, including 1957 up-regulated and 222 down-regulated genes. These genes are mainly annotated in functions such as peptidase activity (especially serine protease activity), protein folding, protein assembly, and cell component assembly, and enriched in pathways such as protein processing in endoplasmic reticulum, lysosome, glutathione metabolism, and sphingolipid metabolism. In addition, some proteins that are closely related to ovarian development, including heat shock protein 70, protein disulfide isomerase, paramyosin, troponin I, hexosaminidase, serine protease, Kunitz serine protease inhibitors, and vitellogenin, were obtained. Conclusions: These findings fill the gap in the biological data for the ovarian development of soft ticks, provide a reference database for subsequent proteomics research, and offer fundamental support for the screening and development of candidate antigens for anti-tick vaccines.

Keywords: Argas persicus; differentially expressed genes; ovary; transcriptome.

Figure 6

Verification of the expression of DEGs at the three time points of two key stages in A. persicus. (A–G) presents the qPCR (left: β-Actin as internal control; middle: GAPDH as internal control) and RNA-seq (right) analyses for the selected seven genes: paramyosin, fatty acid-binding protein, vitellogenin, heat shock protein 70, chitinase, aquaporin, and ferritin heavy-chain. β-Actin (left) and GAPDH (middle) were utilized as internal reference genes. The left Y-axis indicates the relative gene expression levels derived from the qRT-PCR analysis, while the right Y-axis displays the FPKM values obtained from RNA sequencing. The X-axis represents ovary samples at different time points. The error bars indicate the standard deviation of three biological replicates.

Figure 1

Volcano maps and hierarchical clustering analysis showing the expression profiles of genes between OV1 and OV0 (A,B), as well as OV2 and OV1 (C,D), in A. persicus. In the volcano plots (A,B), red dots represent upregulated genes, green dots indicate downregulated genes, and blue dots represent genes with no significant differences. The differentially expressed genes were selected using the threshold padj<0.05 and| log2(foldchange)| > 1.

Figure 2

Gene ontology (GO) enriched terms associated with the DEGs in OV1 vs. OV0 (A,B) and OV2 vs. OV1 (C,D) of ovaries from A. persicus. (A,C) Up-regulated DEGs, (B,D) down-regulated DEGs. The top 20 most significant GO terms were illustrated for each compared pair. The mark symbol * indicates significantly enriched GO terms assigned to the differentially expressed transcripts (padj < 0.05). The X-axis represents the −log10 (p-value) of the enrichment analysis. The Y-axis indicates the GO terms, with “n” in parentheses representing the number of enriched genes.

Figure 3

Statistics of the top 20 KEGG pathways for the DEGs in OV1 vs. OV0 (A,B) and OV2 vs. OV1 (C,D) of ovaries from A. persicus. (A,C) Up-regulated DEGs, (B,D) down-regulated DEGs. The top 20 most significant KEGG terms were illustrated for each compared pair. The mark symbol * indicates significantly enriched KEGG terms assigned to the differentially expressed transcripts (padj < 0.05). The X-axis represents the −log10 (p-value) of the enrichment analysis. The Y-axis indicates the KEGG pathway, with “n” in parentheses representing the number of enriched genes.

Figure 4

Potentially functional genes compose the interactive network between OV1 and OV0 ovaries. The red and green represent up-regulation and down-regulation, respectively. Larger node sizes indicate higher degree values.

Figure 5

Potentially functional genes compose the interactive network between OV2 and OV1 ovaries. The red and green represent up-regulation and down-regulation, respectively. Larger node sizes indicate higher degree values.