Role of nuclear protein Akirin in the modulation of female reproduction in Nilaparvata lugens (Hemiptera: Delphacidae)

Abstract

Introduction: Akirin as a highly conserved transcription factor, exerts a profound influence on the growth, development, immune response, and reproductive processes in animals. The brown planthopper (BPH), Nilaparvata lugens, a major pest in rice production in Asia, possesses high reproductive capacity, a critical factor contributing to reduced rice yields. The aims of this study were to demonstrate the regulatory role of Akirin in the reproduction of BPH. Methods: In this study, quantitative PCR (qPCR) was used to detect the mRNA expression of genes. RNA interference (RNAi) was used to downregulate the expression of Akirin gene, and RNA sequencing (RNA-seq) was used to screen for differentially expressed genes caused by Akirin downregulation. Hormone contents were measured with the enzyme linked immunosorbent assay (ELISA), and protein content was evaluated with the bicinchoninic acid (BCA) method. Results: Using BPH genome data, we screened for an Akirin gene (NlAkirin). An analysis of tissue-specific expressions showed that NlAkirin was expressed in all tissues tested in female BPH, but its expression level was highest in the ovary. After inhibiting the mRNA expression of NlAkirin in BPH females, the number of eggs laid, hatching rate, and number of ovarioles decreased. Transcriptome sequencing was performed, following a NlAkirin double-stranded RNA treatment. Compared with the genes of the control, which was injected with GFP double-stranded RNA, there were 438 upregulated genes and 1012 downregulated genes; the expression of vitellogenin (Vg) and vitellogenin receptor (VgR) genes as well as the mRNA expression of genes related to the target of rapamycin (TOR), juvenile hormone (JH), and insulin pathways involved in Vg synthesis was significantly downregulated. As a result of NlAkirin knockdown, the titers of JH III and Ecdysone (Ecd) were downregulated in unmated females but returned to normal levels in mated females. The ovarian protein contents in both unmated and mated females were downregulated. Discussion and conclusion: Our results suggest that NlAkirin affects female BPH reproduction by regulating the mRNA expression of genes related to the Vg, VgR, TOR, JH, and insulin signaling pathways, in addition to the titers of JH III and Ecd. The findings of this research provide novel insights into the regulatory role of Akirin in insect reproductive capacity.

Keywords: Akirin; Nilaparvata lugens; hormone; reproduction; vitellogenin.

FIGURE 1

Sequence alignment and tissue expression profile analysis of insect Akirin. (A) Akirin protein sequence alignment for insects. The conserved nuclear localization signal (NLS) is shaded blue. (B) Phylogenetic tree of insect Akirin based on amino acid sequences. Phylogenetic tree constructed using the neighbor-joining method. The numbers on the branches of the phylogenetic tree represent bootstrap values from 1000 replicates, and the scale represents genetic distances. The red color represents NlAkirin from the BPH, and the GenBank IDs of each species are as follows: AmAkirin (Apis mellifera, XP_395252.2), SiAkirin (Solenopsis invicta, XP_011169583.1), AeAkirin (Acromyrmex echinatior, XP_011066641.1), SfAkirin (Sogatella furcifera, MG744348), NLAkirin (Nilaparvata lugens, AWT86615.1), RpAkirin (Riptortus pedestris, BAN21089), AgAkirin (Anopheles gambiae, XP_308938.4), CqAkirin (Culex quinquefasciatus, XP_001863200.1), AaAkirin (Aedes albopictus, ACF49499.1), DmAkirin (Drosophila melanogaster, NP_648113.1), TcAkirin (Tribolium castaneum, XP_971340), ObAkirin (Ooceraea biroi, EZA59122.1), BmAkirin (Bombyx mori, NP_001243977.1), and PaAkirin (Pararge aegeria, JAA80553.1). (C) Expression levels of NlAkirin in different tissues of BPH females. Statistical analysis was performed via multiple comparison. Data are shown as mean ± SE of three independent experiments. a, b, c, and d: Turkey test, P < 0.05. The presence of identical letters indicates no significant difference between these two sets of data.

FIGURE 2

Effect of NIAkirin on the reproduction of BPH females. (A) mRNA expression level of NIAkirin after RNAi. (B) Number of total eggs laid. (C) Hatching rate of eggs. (D) Number of ovarioles in the ovary. (E) Representative images of the number of ovarioles, scale bar represents 100 μm. (F) Proportion of mortality due to the large accumulation of eggs in abdomens. (G) Representative images of accumulation of eggs in abdomens, scale bar represents 1 mm. Statistical analysis was performed via the t-test. Data are shown as the mean ± SE of three independent experiments. *P < 0.05; ns, non-significant difference.

FIGURE 3

Analysis of RNA-seq results after dsAkirin injection. (A) Volcano plot of DEGs. (B) Cluster heatmap of DEGs. G1–G3, the three replicates of dsGFP treatment; A1–A3, the three replicates of dsAkirin treatment; The left side depicts the clustering of different genes. The number displayed on the right indicates the level of gene expression. (C) The average FPKM of each gene. The gene ID numbers for Vg1 to Vg4 were 111061268, 111057493, 111061289, and 111061279, respectively. * padj≤0.05 and |log2FoldChange|≥1.0. (D) Cluster heatmap of genes related to female insect reproduction in transcriptome sequencing results; The left side depicts the clustering of different genes. The number displayed on the right indicates the level of gene expression.

FIGURE 4

mRNA expression of Vg, VgR, and related pathway genes. Statistical analysis was performed using the t-test. Data are shown as the mean ± SE of three independent experiments. The gene ID numbers for Vg1 to Vg4 were 111061268, 111057493, 111061289, and 111061279, respectively. *P < 0.05; ns, non-significant difference.

FIGURE 5

Titer results for levels of JH III and Ecd in BPH females treated with dsAkirin. (A) JH III titer in unmated females following dsAkirin injection. (B) Ecd titer in unmated females after dsAkirin injection. (C) JH III titer in mated females after dsAkirin injection and mating with untreated males. (D) Ecd titer in mated females after dsAkirin injection and mating with untreated males. Statistical analysis was performed using the t-test. Data are shown as the mean ± SE of three independent experiments. *P < 0.05; ns, non-significant difference.

FIGURE 6

Protein content in the ovary of BPH females treated with dsAkirin. (A) Ovarian protein content in unmated females following injection of dsAkirin. (B) Ovarian protein content in females after mating with untreated males after injection of dsAkirin. Statistical analysis was performed using a t-test. Data are shown as mean ± SE of three independent experiments. *P < 0.05.