Reduction in Gonad Development and Sperm Motility in Male Brown Planthopper Nilaparvata lugens via RNAi-Mediated Knockdown of tramtrack

Abstract

The brown planthopper Nilaparvata lugens, a major rice pest, threatens global food security through rapid reproduction. This study investigates the role of the tramtrack (ttk) gene in male reproductive development and spermatogenesis using RNA interference (RNAi). Gene expression analysis revealed higher ttk levels in testes. RNAi-mediated knockdown of ttk in fourth-instar male nymphs reduced its expression by up to 80%, leading to severely impaired gonad development. Testes, vas deferens, and accessory glands in treated males exhibited 8-89% volume reductions compared to controls, accompanied by a 51-69% decline in sperm count and 60-85% reduction in sperm motility. Consequently, eggs fertilized by treated males showed a 73% decrease in hatching rates, with arrested embryonic development. These findings demonstrate ttk's critical role in spermatogenesis and gonad maturation in N. lugens, highlighting its potential as an RNAi target for sustainable pest control strategies.

Keywords: Nilaparvata lugens; RNAi; gonad maturation; spermatogenesis; tramtrack.

Figure 1

ttk expression in body regions and male reproductive organs of N. lugens within 24 h after eclosion. Data are expressed as mean ± SD (n = 24), and the analysis method is one-way ANOVA. The different lowercase letters show significant difference (p < 0.05).

Figure 2

RNAi efficiency of dsttk in fourth-instar male nymphs. Data are expressed as mean ± SD (n = 15), and the analysis method is two-tailed Student’s t test. The different lowercase letters show significant difference (p < 0.05).

Figure 3

The hatching rate of eggs laid by female adults mated with the male adults grown from test male nymphs. “control” means untreated samples. Data are expressed as mean ± SD (n = 15), and the analysis method is one-way ANOVA. The different lowercase letters show significant difference (p < 0.05).

Figure 4

The reproductive glands of male adults grown from fourth-instar nymphs with different treatments. (A1–A8) showed the reproductive glands from adults grown from untreated fourth-instar nymphs on day 1 to 8 post-eclosion. (B1–B8) showed the reproductive glands of adults grown from fourth-instar nymphs treated with dsGFP on day 1 to 8 post-eclosion. (C1–C8) showed the reproductive glands of adults grown from fourth-instar nymphs treated with dsttk on day 1 to 8 post-eclosion. Boxes: testes (red), vas deferens (green), and accessory glands (blue). Scale bar: 500 μm.

Figure 5

Trends in testis volume (A), vas deferens volume (B), and accessory gland volume (C) from days 1 to 8 of brown planthopper male adults grown from fourth-instar nymphs with different treatments. Data are expressed as mean ± SD (n = 9), and the analysis method is one-way ANOVA, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Trends in total sperm count (A), proportion of motile sperm (B), motility frequency of motile sperm (C), and motility amplitude of motile sperm (D) from days 1 to 8 post-eclosion of brown planthopper male adults grown from fourth-instar nymphs with different treatments. Data are expressed as mean ± SD (n = 9), and the analysis method is one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001.