RNA interference of a trehalose-6-phosphate synthase gene reveals its roles in the biosynthesis of chitin and lipids in Heortia vitessoides (Lepidoptera: Crambidae)

Abstract

Trehalose-6-phosphate synthase (TPS), an enzyme that hydrolyzes two glucose molecules to yield trehalose, plays a pivotal role in various physiological processes. In this study, we cloned the trehalose-6-phosphate synthase gene (HvTPS) and investigated its expression patterns in various tissues and developmental stages in Heortia vitessoides Moore (Lepidoptera: Crambidae). HvTPS was highly expressed in the fat body and after pupation or before molting. We knocked down TPS in H. vitessoides by RNA interference and found that 3.0 μg of dsHvTPS resulted in optimal interference at 24 h and 36 h post-injection and caused a sharp decline in the survival rate during the 5th instar larval-pupal stage and obviously abnormal or lethal phenotypes. Additionally, compared to the controls, TPS activity and trehalose contents were significantly lower and the glucose content was significantly higher 24 h or 36 h after injection with 3.0 μg of dsHvTPS. Furthermore, the silencing of HvTPS suppressed the expression of six key genes in the chitin biosynthesis pathway and one key gene related to lipid catabolism. The expression levels of two genes associated with lipid biosynthesis were upregulated. These results strongly suggest that HvTPS is essential for the normal growth and development of H. vitessoides and provide a reference for further studies of the utility of key genes involved in chitin and lipid biosynthesis for controlling insect development.

Keywords: Heortia vitessoides Moore; RNA interference; chitin biosynthesis; lipid biosynthesis; metamorphosis; trehalose-6-phosphate synthase.

Figure 1

Phylogenetic tree of trehalose phosphate synthases (TPS) in Heortia vitessoides and other insects. The tree was constructed by the neighbor‐joining method using MEGA. The sequences were obtained from GenBank under the following accession numbers: Spodoptera litura (ADA63844), Spodoptera exigua (ABM66814), Helicoverpa armigera (XP_021201246), Bombyx mori (XP_004926812), Amyelois transitella (XP_013187220), Gampsocleis gratiosa (APZ77037), Pogonomyrmex barbatus (XP_011641245), Linepithema humile (XP_012234592), Megachile rotundata (XP_003702415), Apis cerana (XP_016905400) and Apis mellifera (XP_00324923). Scale bar represents 0.02 substitutions per site. Numbers at nodes indicate bootstrap support.

Figure 2

Relative expression levels of HvTPS in different tissues (A) and at different developmental stages (B) of Heortia vitessoides. Head (HE), epidermis (EP), fat body (FB), midgut (MG) and Malpighian tubules (MT). L4–L5, 4th and 5th instar larvae; EP, early pupae; P5 and P10, 5‐day‐old and 10‐day‐old pupae; LP, late pupae; A1–A2, 1‐day‐old and 2‐day‐old adults. Error bars represent the standard error of the calculated means based on three biological replicates. Different letters above the error bars indicate significant differences (P < 0.05).

Figure 3

Changes in the messenger RNA (mRNA) and enzyme activity levels of HvTPS and the glucose or trehalose content after specific RNA interference (RNAi). (A) Relative expression levels of HvTPS in 4th‐instar larvae after injection with dsHvTPS at doses of 1.0, 2.0, 3.0, 4.0 and 5.0 μg/individual. (B) Changes in the glucose content after RNAi. (C) Changes in the trehalose content after RNAi. (D) Changes in HvTPS activity after dsHvTPS injection. Error bars represent the standard error of the calculated means based on three biological replicates. Different letters above the error bars indicate significant differences among treatments and the control measured at the same time (P < 0.05).

Figure 4

Analysis of phenotypes and survival rates after injection with dsHvTPS. (A) During the larval–pupal stage, insects produced three lethal phenotypes: severe‐abnormal, abdomen‐abnormal and half‐eclosion. During the pupal‐adult stage, insects produced one lethal phenotype: misshapen wings. (B) Survival rates after the injection of dsHvTPS. The survival rates of insects after dsHvTPS injections for 4th instar larvae to adults. Error bars represent the standard error of the calculated means based on three biological replicates. Different letters above the error bars indicate significant differences between treatments and controls measured at the same time point (P < 0.05).

Figure 5

Relative expression levels of key genes in the chitin biosynthesis pathway after RNA interference (RNAi). The relative expression levels of HvTRE‐1 (A), HvTRE‐2 (B), HvG6PI (C), HvUAP (D), HvCHS‐1 (E), and HvCHS‐2 (F) in Heortia vitessoides at 24 h and 36 h after injection with 3.0 μg of dsHvTPS. Error bars represent the standard error of the calculated means based on three biological replicates. Different letters on the error bars indicate significant differences (P < 0.05).

Figure 6

Changes in lipids in HvTPS RNA interference (RNAi) larvae. (A) Fat body size and (B) fat body weight after larvae were injected with 3 μg of dsHvTPS and dissected at 72 h. Expression levels of two genes related to fatty acid biosynthesis, (C) acetyl‐CoA carboxylase (HvACC) and (D) fatty acid synthase (HvFAS), and (E) lipase 1 (HvLIP1) associated with lipid degradation at 24 h and 36 h after the knockdown of HvTPS. Error bars represent the standard error of the calculated means based on three biological replicates. Different letters on the error bars indicate significant differences (P < 0.05).