Effect of Insulin Receptor on Juvenile Hormone Signal and Fecundity in Spodoptera litura (F.)

Abstract

Insulin signaling can regulate various physiological functions, such as energy metabolism and reproduction and so on, in many insects, including mosquito and locust. However, the molecular mechanism of this physiological process remains elusive. The tobacco cutworm, Spodoptera litura, is one of the most important pests of agricultural crops around the world. In this study, phosphoinositide 3-kinase (SlPI3K), protein kinase B (SlAKT), target of rapamycin (SlTOR), ribosomal protein S6 kinase (SlS6K) and transcription factor cAMP-response element binding protein (SlCREB) genes, except transcription factor forkhead box class O (SlFoxO), can be activated by bovine insulin injection. Then, we studied the influence of the insulin receptor gene (SlInR) on the reproduction of S. litura using RNA interference technology. qRT-PCR analysis revealed that SlInR was most abundant in the head. The SlPI3K, SlAKT, SlTOR, SlS6K and SlCREB genes were decreased, except SlFoxO, after the SlInR gene knockdown. Further studies revealed that the expression of vitellogenin mRNA and protein, Methoprene-tolerant gene (SlMet), could be down-regulated by the injection of dsRNA of SlInR significantly. Furthermore, a depletion in the insulin receptor by RNAi significantly decreased the content of juvenile hormone III (JH-III), total proteins and triacylgycerol. These changes indicated that a lack of SlInR could impair ovarian development and decrease fecundity in S. litura. Our studies contribute to a comprehensive insight into reproduction, regulated by insulin and the juvenile hormone signaling pathway through nutrition, and a provide theoretical basis for the reproduction process in pest insects.

Keywords: insulin pathway; juvenile hormone; nutrition; reproduction; vitellogenin.

Figure 1

Expression profile of SlInR in different tissues and effect of bovine insulin injection on gene expression of insulin signaling pathway. (A) qPCR analysis of SlInR expression levels in different tissues from one-day-old adult females. (B) The expression of insulin signaling pathway genes was detected 24 h after bovine insulin injection by qRT-PCR. Results are represented as means ± SD of three independent samples and samples are normalized to SlRPL10 expression levels. Different lowercase letters represent significant differences of SlInR levels among various tissues determined by one-way ANOVA. Statistically significant difference between the PBS and bovine insulin injection groups (* p < 0.05; ** p < 0.01, Student’s t-test).

Figure 2

Efficiency of SlInR gene knockdown and effect of SlInR knockdown on gene expression of insulin signaling pathway. (A) Efficiency of SlInR gene knockdown at 12, 24, 36 and 48 h. (B) The expression of insulin signaling pathway genes was detected 24 h after dsSlInR injection by qRT-PCR. Results are represented as means ± SD of three independent samples and samples are normalized to SlRPL10 expression levels (* p < 0.05; ** p < 0.01, Student’s t-test).

Figure 3

Effect of SlInR knockdown on the contents of protein and triglyceride. (A) Protein contents in the abdomen after SlInR knockdown (n = 25). (B) Triglyceride contents in ovaries after SlInR knockdown (n = 25). Statistically significant difference between the dsEGFP and dsInR injection groups (* p < 0.05; ** p < 0.01, Student’s t-test).

Figure 4

Effect of SlInR silence on SlVg expression and reproduction. (A) qRT-PCR analysis of Vg transcript abundance in abdomen from females treated with dsSlInR for 12, 24, 36 and 48 h. (B) Western blotting analysis of SlVg contents in fat body. Immunoblot of fat body collected 48 h from females injected with dsSInR and dsEGFP and detection with Vg antibody, an antibody against actin was used as a loading control. (C) Daily fecundity. The newly emerged female adults were treated with dsRNA and the fecundity of single female was recorded for six consecutive days after mating (n = 25). Statistically significant difference between the dsEGFP and dsInR injection groups (* p < 0.05; ** p < 0.01, Student’s t-test).

Figure 5

Effect of SlInR interference on SlMet expression and JH-III titer. (A) qRT-PCR analysis of SlMet expression levels after SlInR knockdown. (B) JH-III titer was evaluated after SlInR gene knockdown by HPLC. Results are represented as means ± SD of three independent replicates. Statistically significant difference between the dsEGFP and dsInR injection groups (** p < 0.01, Student’s t-test).

Figure 6

Effect of SlInR interference on ovarian development. dsSlInR and dsEGFP were injected after eclosion and the ovaries of female adults were dissected 48 h later. The scale bar represents 1 mm.