Nutritional Signaling Regulates Vitellogenin Synthesis and Egg Development through Juvenile Hormone in Nilaparvata lugens (Stål)

Abstract

Insect female reproduction which comprises the synthesis of vitellogenein (Vg) in the fat body and its incorporation into developing oocytes, needs a large amount of energy and food resources. Our previous studies found that juvenile hormone (JH) regulates vitellogenesis in the brown planthopper, Nilaparvata lugens. Here, we report on the role of JH in nutrient-regulated Vg synthesis and egg development. We first cloned the genes coding for juvenile hormone acid methyltransferase (JHAMT) which is involved in JH biosynthesis and methoprene-tolerant (Met) for JH action. Amino acids (AAs) induced the expression of jmtN, while showing no effects on the expression of met using an artificial diet culture system. Reduction in JH biosynthesis or its action by RNA interference (RNAi)-mediated silencing of jmtN or met led to a severe inhibition of AAs-induced Vg synthesis and oocyte maturation, together with lower fecundity. Furthermore, exogenous application of JH III partially restored Vg expression levels in jmtN RNAi females. However, JH III application did not rescue Vg synthesis in these met RNAi insects. Our results show that AAs induce Vg synthesis in the fat body and egg development in concert with JH biosynthesis in Nilaparvata lugens (Stål), rather than through JH action.

Keywords: Juvenile hormone; Nilaparvata lugens; RNA inteference; nutritional signaling; vitellogenesis.

Figure 1

Phylogenetic analysis of Met (A) and JHAMT (B) from different insect species based on the amino acid sequences alignment. Protein sequences were downloaded from the GenBank database and the accession numbers are shown. The phylogenetic tree was generated by MEGA6 using the Maximum Likelihood method. Bootstrap values (1000 replicates) are shown in the cladogram. The genetic distance is drawn to scale.

Figure 2

Tissue-specific expression profiles of met (A) and jmtN (B). All tissues were dissected from three-day-old adult females. BR: brain; CA: corpora allata; FB: fat body; OV: ovary; MG: Malpighian tubules; EP: epidermis. Values represent the mean of 7–8 independent pools of 30–50 females and three technical replicates, normalized with β-actin (n = 7–8). Vertical bars indicate standard errors. Different lowercase letters above the columns indicate significant differences within different tissues (one-way ANOVA, p < 0.05).

Figure 3

AAs regulate Vg synthesis. (A) Diagram of experimental design. AFE, after adult female emergence. AD3d, three-day-old AAs-deprived females; AF3d, three-day-old AAs-fed females; AD2d, two-day-old AAs-deprived females; AR3d, two-day-old AAs-deprived females were fed on normal diet (+AAs) for another day; (B) Relative expression levels of Vg mRNA in the fat body were detected by qRT-PCR using β-actin as a reference. Reactions were performed based on independent RNA sample preparations and values were shown as mean ± SE (n = 8). Asterisk denoted significant differences from controls (Student’s t-test, “*” denotes p < 0.05). Vg protein levels were detected by western blot, an antibody against β-actin was used as a loading control.

Figure 4

Expression of met (A) and jmtN (B) mRNA in AAs-deprived females. Newly emerged females (within 12 h) were reared on artificial diets either containing (+AAs) or lacking amino acids (−AAs). Relative mRNA expression levels of met in the FB and jmtN in the CA were detected using qRT-PCR. Bars represent mean ± SE of 7–8 biologically independent pools of 30–50 females and three technical replicates, normalized with β-actin (n = 7–8, Student’s t-test, “ns” denotes p > 0.05 and “*” denotes p < 0.05).

Figure 5

Knockdown of met or jmtN by RNAi results in significant downregulation of Vg induced by AAs. (A) Knockdown efficiency of met and jmtN by RNAi. Relative mRNA expression levels of met in the FB and jmtN in the CA in gfp dsRNA control females were set to 1; (B) Effect of the JH pathway genes knockdown on the mRNA and protein levels of Vg. Total RNA and protein were isolated from fat bodies of females. Vg transcript levels were detected in comparison to β-actin mRNA levels by qRT-PCR. The expression levels of Vg transcripts in each RNAi treatment were determined by setting the control insects (gfp dsRNA) Vg transcript expression levels at 1. Values were shown as mean ± SE of seven independent replications (n = 7). Asterisk denotes significant differences from controls (Student’s t-test, “*” denotes p < 0.05). Vg levels were analyzed by western blot and β-actin was used as a loading control.

Figure 6

Effect of JH III on AAs-induced Vg synthesis. Newly emerged females were injected with 100 ng of respective dsRNA targeting met, jmtN or a nonspecific gfp and reared for two days. After that, the insects were topically applied with JH III (100 ng in 100 nL acetone) or acetone (100 nL) and fed with artificial diets for another day. Relative mRNA levels of Vg in the FB were detected by qRT-PCR (n = 7, Student’s t-test, “ns” denotes p > 0.05 and “*” denotes p < 0.05). Vg protein levels were detected by western blot.

Figure 7

Ovarian growth and fecundity in the RNAi, AAs-deprived and fed females. (A) AAs, met and jmtN are required for ovary development. Equal amount of dsRNA for JH pathway genes and gfp (control) was injected into newly emerged females (within 12 h). The ovaries were dissected on day 6 post adult emergence and photographed with stereo microscopy SMZ18 (Nikon, Tokyo, Japan). Scale bars indicate 500 μm; (B) Fecundity was reduced in met and jmtN dsRNA-treated females. The total number of eggs laid was counted for fifteen days. Since AAs-deprived females laid no eggs, only egg numbers derived from AAs-fed females were used for statistical test. Bars represent mean ± SE of three independent experiments of ten females and asterisk indicates significant differences (Student's t-test, “*” denotes p < 0.05).