Role of Groucho and Groucho1-like in Regulating Metamorphosis and Ovary Development in Nilaparvata lugens (Stål)

Abstract

Juvenile hormone and ecdysone are key regulators in the metamorphosis and development. Grocho (Gro) is a highly conserved protein required for metamorphosis and development. Brown planthopper (Nilaparvata lugens) is a major pest affecting rice production in China and many Asian countries. Although the molecular function of Gro has been investigated in holometabolous insects such as Aedes aegypti and Drosophila melanogaster, their role in the hemimetabolous insect, brown planthopper, and the relationship between NlGro/NlGro1-L and JH/ecdysone signaling pathway, remained unknown. In this study, NlGroucho (NlGro) and NlGroucho1-like (NlGro1-L) were cloned. An analysis of the predicted protein sequence showed that NlGro has highly conserved Q domain and WD40 domain, and NlGro1-L has a highly conserved WD40 domain. The expression profiles of both genes were studied by quantitative real-time PCR (qRT-PCR). Their relative expressions were high in egg, head, wing, ovary, and testis. NlGro and NlGro1-L were found to interact genetically with juvenile hormone and ecdysone signaling by hormone treatment and RNAi of JH/ecdysone signaling-related genes. Moreover, when NlGro or NlGro1-L was down-regulated alone, the survival rate was decreased, the ovarian development was delayed, and the oviposition was also affected. All defects were aggravated when NlGro and NlGro1-L were down-regulated together. This study will help to develop new pesticides on the basis of the function of NlGro and NlGro1-L, and provide new possibilities for the control of Nilaparvata lugens.

Keywords: Ecdysone; Nilaparvata lugens; NlGroucho; NlGroucho1-like; juvenile hormone.

Figure 1

Alignment of NlGro and NlGro1-L.

Figure 2

Expression profiles of NlGro and NlGro1-L. (A) Expression of NlGro at different embryonic stages. (B) Expression of NlGro1-L at different embryonic stages. (E) Embryo; 1–9 day(s) after eggs were laid. (C) Expression of NlGro at different developmental stages. (D) Expression of NlGro1-L at different developmental stages. Egg: egg; 1st to 5th: first to fifth instar nymph; LWF: long-winged female, LWM: long-winged male, SWF: short-winged female, SWM: short-winged male. (E): Expression of NlGro1 in different tissues. (F) expression of NlGro1-L in different tissues. Br: Brain; Th: Thorax; Wi: forewing; Leg: Leg; Mi: mid-gut; Ov: Ovary; Ts: testis; LWF: long-winged female, LWM: long-winged male, SWF: short-winged female, SWM: short-winged male. One-way analysis of variance (Duncan’s method) was used. For the letters a–h, the same letter indicates that there is no significant difference, and different letters indicate a significant difference between the two; the farther the letter is, the greater the difference between the two, p < 0.05.

Figure 3

Expressions of NlGro and NlGro1-L were regulated by JH and ecdysone. (A) The effect of JHIIIon the expression of NlGro and NlGro1-L. Acetone: control; Methoprene: a JH analogue; JHIII: Juvenile Hormone III. (B) The effect of ecdysone on the expression of NlGro and NlGro1-L. Acetone: control; 0.05 ED: 0.05, ecdysone; 0.1 ED: 0.1, ecdysone. (C) Changes in the expression of NlGro after hormone-related genes are down-regulated. (D) Changes in the expression of NlGro1-L after hormone-related genes are down-regulated. One-way analysis of variance (Duncan’s method) was used. The same letter indicates that there is no significant difference, and different letters indicate a significant difference, p < 0.05.

Figure 4

Molting disruption by down-regulating NlGro and NlGro1-L. (A–F) normal and disrupted adult emerged after injection of dsRNA: (A) Control: dsGFP; (B), dsNlGro; (C), dsNlGro1-L; (D), dsNlE93; (E), dsNlGro+dsN1E93; (F), dsNlGro1-L+dsNlE93. (G) Percent of survival of brown planthoppers injected with NlGro and NlGro1-L. Duncan’s multiple comparison was used. The letters a, b, and c indicate whether there is a difference in statistics: the same letter indicates that there is no difference, while different letters indicate significant differences, p < 0.05. (H) Percent of ecdysis failure and survival after injection of dsNlGro. (I) Percent of ecdysis failure and survival after injection of dsNlGro-L.

Figure 5

Ovarian development changed after down-regulation of NlGro and NlGro1-L. Ovarian grade (I) (dsNlGro, dsNlGro-L, dsNlGro + dsNlGro-L), (II) (dsGFP, dsNlGro, dsNlGro-L, dsNlGro + dsNlGro-L), (III) (dsGFP, dsNlGro, dsNlGro-L, dsNlGro + dsNlGro-L), and (IV) (dsGFP, dsNlGro, dsNlGro-L, dsNlGro + dsNlGro-L) are shown.

Figure 6

Down-regulation of NlGro and NlGro1-L affected ovary development and Vg expression. (A) Pre-oviposition period after down-regulation of NlGro and NlGro1-L; Duncan’s multiple comparison was used. The letters a and b indicate whether there is a statistical difference; the same letter represents no difference, while different letters represent significant differences, p < 0.05. (B) Ovarian grading after down-regulating NlGro and NlGro1-L; independent sample t test was used (Student’s t test), n.s.: no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001. (C) The total number of eggs reduced after down-regulating NlGro and NlGro1-L; Duncan’s multiple comparison was used. The letters a and b indicate whether there is a statistical difference; the same letter represents no difference, while different letters represent significant differences, p < 0.05. (D) Relative expression of Vg after the down-regulation of NlGro and NlGro1-L; Duncan’s multiple comparison was used. The letters a and b indicate whether there is a statistical difference; the same letter represents no difference, while different letters represent significant differences, p < 0.05. (E,F) Western blot detection of Vg protein after down-regulation of NlGro and NlGro1-L: (E) Abdomen, (F) Ovary.

Figure 7

Model of Gro and Gro1-L involved in JH and the ecdysone signaling pathway.