Nuclear receptor gene, HR3 and βFTZ-F1, involved in molting development, pupation and eclosion in Hyphantria cunea (Drury）

Published Apr 29, 2026 on Dryad. https://doi.org/10.5061/dryad.z34tmpgv0

Data files

Apr 29, 2026 version files 121.73 KB

doi_10_5061_dryad_z34tmpgv0.zip

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README.md

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Abstract

Nuclear receptor genes play a crucial role in insect molting. These genes, found in many insects, produce important proteins that control the expression of other genes, which in turn regulate the molting process in larvae. This study investigates the roles of nuclear receptor genes HcHR3 and HcβFTZ-F1in the molting development, pupation and eclosion of Hyphantria cunea. The expression levels of two gene mRNAs were detected in all developmental stages and various adult tissues. The novel pET28a (+)-HT115 (DE3) RNase III-system was successfully constructed to express HcHR3 and HcβFTZ-F1-dsRNA against H. cunea efficiently. To explore the physiological functions of these genes, they were knocked down in larvae and pupae using dsRNA extracted from bacteria, resulting in a decrease in the expression levels of HcHR3 and HcβFTZ-F1, which were decreased by 13.70% to 99.01%. Molting defects were observed in H. cunea larvae following HcHR3 and HcβFTZ-F1 silencing, with a phenotypic percentage ranging from 33.40% to 45.22%. Additionally, knockdown of the two genes in fifth instar larvae decreased weight, food intake and survival rate, resulting in abnormal development during the larval–pupal transition. RNAi-mediated knockdown of HcHR3 and HcβFTZ-F1 prevented the eclosion of the pupae, with malformation rates ranging from 75.67% to 80.12%, and epidermis tanning defects were observed in pupae or adults. Our findings demonstrate that HcHR3 and HcβFTZ-F1 are indispensable for normal post-embryonic development, and they provide validated molecular targets for RNAi-based pest management and insight into outbreak mechanisms of H. cunea.

Dataset DOI: 10.5061/dryad.z34tmpgv0

Description of the data and file structure

Temporal and tissue-based expression profiles of HcHR3 and HcβFTZ-F1.

Effects of dsHcHR3 and dsHcβFTZ-F1 RNAi on larval development and molting.

Effects of dsHcHR3 and dsHcβFTZ-F1 RNAi on pupation and eclosion.

Files and variables

Raw_data_for-Figure2-Relative-expression-level-of-Hchr3-development-stage.xlsx

Data brief description: In Figure 2A, we sampled Hyphantria cunea individuals at different developmental stages: egg stage, 1st to 7th instar larvae (1–3 days old), female pupae on day 1, male pupae on day 1, female adults on day 1, and male adults on day 1. Quantitative real-time PCR (qRT-PCR) was performed to assess changes in HcHR3 expression levels. Each treatment had three mechanical and three biological replicates. The ct value is the original data detected by the instrument. After calculation by the formula, 2^-∆∆Ct is obtained. We calculated the relative expression of the target gene according to the 2^-∆∆Ct. We selected EF-1a and RPL13 as internal reference genes. The expression levels are shown relative to those in the egg stage.

Ct: The number of threshold cycles, the number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold.

2^-∆∆Ct: The gene expression level change of the treatment group relative to the control group.

The naming of different treatments is as follows: Egg, 1st – 7th instar larvae (1L-7L), 1st-3rd day (D1-D3), female pupae (FP), male pupae (MP), female adult (FA), and male adult (MA).

Raw_data_for-Figure2-Relative-expression-level-of-Hcftz-f1-development-stage.xlsx

Data brief description: In Figure 2B, we sampled Hyphantria cunea individuals at different developmental stages: egg stage, 1st to 7th instar larvae (1–3 days old), female pupae on day 1, male pupae on day 1, female adults on day 1, and male adults on day 1. Quantitative real-time PCR (qRT-PCR) was performed to assess changes in HcβFTZ-F1 expression levels. Each treatment had three mechanical and three biological replicates. The ct value is the original data detected by the instrument. After calculation by the formula, 2^-∆∆Ct is obtained. We calculated the relative expression of the target gene according to the 2^-∆∆Ct. We selected EF-1a and RPL13 as internal reference genes. The expression levels are shown relative to those in the egg stage.

The naming of different treatments is as follows: Egg, 1st – 7th instar larvae (1L-7L), 1st-3rd day (D1-D3), female pupae (FP), male pupae (MP), female adult (FA), and male adult (MA).

Raw_data_for-Figure2-Relative-expression-level-of-HcHR3-tissue.xlsx

Data brief description: In Figure 2C，samples were collected from the head, epidermis, fat body, foregut, midgut, hindgut, silk gland, Malpighian tube testis and ovary of 7th instar H. cunea larvae starved for 12 h. Quantitative real-time PCR (qRT-PCR) was performed to assess changes in HcHR3 expression levels. Each treatment had three mechanical and three biological replicates. The ct value is the original data detected by the instrument. After calculation by the formula, 2^-∆∆Ct is obtained. The bars represent 2^−ΔΔCT values (± SEs) normalized to the expression of two internal control genes (EF-1α and RPL13).The expression levels are shown relative to those in the fat body tissue.

Raw_data_for-Figure2-Relative-expression-level-of-Hcftz-f1-tissue.xlsx

Data brief description: In Figure 2D，samples were collected from the head, epidermis, fat body, foregut, midgut, hindgut, silk gland, Malpighian tube testis and ovary of 7th instar H. cunea larvae starved for 12 h. Quantitative real-time PCR (qRT-PCR) was performed to assess changes in HcβFTZ-F1 expression levels. Each treatment had three mechanical and three biological replicates. The ct value is the original data detected by the instrument. After calculation by the formula, 2^-∆∆Ct is obtained. The bars represent 2^−ΔΔCT values (± SEs) normalized to the expression of two internal control genes (EF-1α and RPL13).The expression levels are shown relative to those in the fat body tissue.

Raw_data_for-Figure4-Silencing-efficiency-of-HcHR3-or-HcβFTZ-F1-5th.xlsx

Data brief description: In Figure 4A，larvae were injected once with dsRNA on the first day of the 5th instar. Samples were collected at 48 h and 72 h post-injection to evaluate the RNAi silencing efficiency of HcHR3 and HcβFTZ-F1. Larvae injected with dsGFP at corresponding time points served as the control. Data are presented as mean ± standard error (SE) (n = 3). Quantitative real-time PCR (qRT-PCR) was performed to assess changes in HcβFTZ-F1 expression levels. Each treatment had three mechanical and three biological replicates. The ct value is the original data detected by the instrument. After calculation by the formula, 2-∆∆Ct is obtained. We calculated the relative expression of the target gene according to the 2-∆∆Ct. We selected EF-1a and RPL13 as internal reference genes. The expression levels are shown relative to those in dsGFP at the same point. Statistical significance of gene expression differences between the two groups was calculated using Student's t-test (ns, P > 0.05; * P < 0.05; P < 0.01; *** P < 0.001; ** P < 0.0001).

dsHcHR3-48h: 5th instar larvae in the experimental group at 48 hours after injection of dsRNA targeting the HcHR3 gene.

dsHcHR3-72h: 5th instar larvae in the experimental group at 72 hours after injection of dsHcHR3.

dsGFP-48h: 5th instar larvae in the control group at 48 hours after injection of the negative control dsGFP.

dsGFP-72h: 5th instar larvae in the control group at 72 hours after injection of dsGFP.

dsHcβFTZ-F1-48h: 5th instar larvae in the treatment group at 48 hours after injection of dsRNA targeting the HcβFTZ-F1 gene.

dsHcβFTZ-F1-72h: 5th instar larvae in the treatment group at 72 hours after injection of dsHcβFTZ-F1.

dsGFP-48h: 5th instar larvae in the control group at 48 hours after injection of the negative control dsGFP.

dsGFP-72h: 5th instar larvae in the control group at 72 hours after injection of dsGFP.

Raw_data_for-Figure4-Survival-rates-of-larvae.xlsx

Data brief description: In Figure 4B，using injection of 2 μg dsGFP as a negative control, 2 μg of dsHcHR3 or 2 μg of dsHcβFTZ-F1 were injected into first-day 5th instar larvae of H. cunea. Survival rates of larvae in each treatment group and the control group were recorded at 24, 48, 72, and 96 hours post-injection, and survival curves were plotted accordingly (0 represents surviving individuals, 1 represents dead individuals). Each treatment consisted of 30 larvae and was independently replicated three times. The log‑rank (Mantel‑Cox) test was used to analyze the significance of differences among the survival curves (ns P>0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

The following double-stranded RNA (dsRNA) constructs were injected as silencing agents into first-day 5th instar larvae of H. cunea:

dsGFP (negative control): targeting the green fluorescent protein (GFP) gene. Injection of dsGFP does not interfere with the expression of endogenous genes in H. cunea and is used to exclude non‑specific effects caused by the injection procedure or the dsRNA itself.

dsHcHR3: targeting the HcHR3 gene of H. cunea.

dsHcβFTZ‑F1: targeting the HcβFTZ‑F1 gene of H. cunea.

Raw_data_for-Figure4-Body-weight-and-food-intake-of-larvae.xlsx

Data brief description: In Figure 4C, fifth-instar (day 1) larvae of H. cunea were microinjected with 2 μg of dsRNA solution (dsGFP, dsHcHR3, or dsHcβFTZ‑F1) into the penultimate posterior abdominal segment. Fresh mulberry leaves were provided daily, and the rearing containers were cleaned each day. Larval body weight and food intake were recorded every 24 hours for a total of 72 hours. Food intake was calculated using the following formula: food intake = leaf mass before feeding − leaf mass after feeding. To minimize the effect of leaf water evaporation on the experimental data, the size of the mulberry leaves added each time was carefully controlled. Both the leaves and larval body weight were weighed..Different letters indicate significant differences (P < 0.05, one-way ANOVA, Tukey’s test). Each point represents the mean ± SEM from three independent experiment.

Units: body weight and food intake in mg; time points in days post-treatment.

dsGFP Food intake (24h, 48h, 72h): Larval food intake measured at 24, 48, and 72 hours after injection of the negative control dsGFP.

dsHcHR3 Food intake (24h, 48h, 72h): Larval food intake measured at 24, 48, and 72 hours after injection of dsHcHR3.

dsHcβFTZ‑F1 Food intake (24h, 48h, 72h): Larval food intake measured at 24, 48, and 72 hours after injection of dsHcβFTZ‑F1.

dsGFP Body weight (24h, 48h, 72h): Larval body weight measured at 24, 48, and 72 hours after injection of the negative control dsGFP.

dsHcHR3 Body weight (24h, 48h, 72h): Larval body weight measured at 24, 48, and 72 hours after injection of dsHcHR3.

dsHcβFTZ‑F1 Body weight (24h, 48h, 72h): Larval body weight measured at 24, 48, and 72 hours after injection of dsHcβFTZ‑F1.

Raw_data_for-Figure4-phenotype-rates-of-larvae.xlsx

Data brief description: In Figure 4D, phenotypes and phenotype rates of larvae injected with dsHcHR3 and dsHcβFTZ-F1. dsGFP shows normal moulting as a control (n=30). dsHcHR3 and dsHcβFTZ-F1 resulted in abnormal moulting or nonmoulting. The dsHcHR3 and dsHcβFTZ-F1 exhibit moulting difficulties, characterized by the inability to shed their old cuticles completely. The phenotypic rate was analysed using Fisher's exact test.

Raw_data_for-Figure5-Silencing-efficiency-of-HcHR3-or-HcβFTZ-F1-7th.xlsx

Data brief description: In Figure 5A，larvae were injected once with dsRNA on the first day of the 7th instar. Samples were collected at 48 h and 72 h post-injection to evaluate the RNAi silencing efficiency of HcHR3 and HcβFTZ-F1. Larvae injected with dsGFP at corresponding time points served as the control. Data are presented as mean ± standard error (SE) (n = 3). Quantitative real-time PCR (qRT-PCR) was performed to assess changes in HcβFTZ-F1 expression levels. Each treatment had three mechanical and three biological replicates. The ct value is the original data detected by the instrument. After calculation by the formula, 2^-∆∆Ct is obtained. We calculated the relative expression of the target gene according to the 2^-∆∆Ct. We selected EF-1a and RPL13 as internal reference genes. The expression levels are shown relative to those in dsGFP at the same point. Statistical significance of gene expression differences between the two groups was calculated using Student's t-test (ns, P > 0.05; * P < 0.05; P < 0.01; *** P < 0.001; ** P < 0.0001).

dsHcHR3-48h: 7th instar larvae in the experimental group at 48 hours after injection of dsRNA targeting the HcHR3 gene.

dsHcHR3-72h: 7th instar larvae in the experimental group at 72 hours after injection of dsHcHR3.

dsGFP-48h: 7th instar larvae in the control group at 48 hours after injection of the negative control dsGFP.

dsGFP-72h: 7th instar larvae in the control group at 72 hours after injection of dsGFP.

dsHcβFTZ-F1-48h: 7th instar larvae in the treatment group at 48 hours after injection of dsRNA targeting the HcβFTZ-F1 gene.

dsHcβFTZ-F1-72h: 7th instar larvae in the treatment group at 72 hours after injection of dsHcβFTZ-F1.

dsGFP-48h: 7th instar larvae in the control group at 48 hours after injection of the negative control dsGFP.

dsGFP-72h: 7th instar larvae in the control group at 72 hours after injection of dsGFP.

Raw_data_for-Figure5-Silencing-efficiency-of-HcHR3-or-HcβFTZ-F1-prepupa.xlsx

Data brief description: In Figure 5B，larvae were injected once with dsRNA on the first day of the prepupal stage. Samples were collected at 48 h and 72 h post-injection to evaluate the RNAi silencing efficiency of HcHR3 and HcβFTZ-F1.Prepupa s injected with dsGFP at corresponding time points served as the control. Data are presented as mean ± standard error (SE) (n = 3). Quantitative real-time PCR (qRT-PCR) was performed to assess changes in HcβFTZ-F1 expression levels. Each treatment had three mechanical and three biological replicates. The ct value is the original data detected by the instrument. After calculation by the formula, 2^-∆∆Ct is obtained. We calculated the relative expression of the target gene according to the 2^-∆∆Ct. We selected EF-1a and RPL13 as internal reference genes. The expression levels are shown relative to those in dsGFP at the same point. Statistical significance of gene expression differences between the two groups was calculated using Student's t-test (ns, P > 0.05; * P < 0.05; P < 0.01; *** P < 0.001; ** P < 0.0001).

dsHcHR3-48h: Prepupa in the experimental group at 48 hours after injection of dsRNA targeting the HcHR3 gene.

dsHcHR3-72h: Prepupa in the experimental group at 72 hours after injection of dsHcHR3.

dsGFP-48h: Prepupa in the control group at 48 hours after injection of the negative control dsGFP.

dsGFP-72h:Prepupa in the control group at 72 hours after injection of dsGFP.

dsHcβFTZ-F1-48h: Prepupa in the treatment group at 48 hours after injection of dsRNA targeting the HcβFTZ-F1 gene.

dsHcβFTZ-F1-72h:Prepupa in the treatment group at 72 hours after injection of dsHcβFTZ-F1.

dsGFP-48h: Prepupa in the control group at 48 hours after injection of the negative control dsGFP.

dsGFP-72h: Prepupa in the control group at 72 hours after injection of dsGFP.

Raw_data_for-Figure5-phenotype-rates-of-pupa.xlsx

Data brief description: In Figure 5C, phenotypes and phenotype rates of pupa injected with dsHcHR3 and dsHcβFTZ-F1. dsGFP shows normal moulting as a control (n=30). dsHcHR3 and dsHcβFTZ-F1 resulted in abnormal moulting or nonmoulting. The dsHcHR3 and dsHcβFTZ-F1 exhibit moulting difficulties, characterized by the inability to shed their old cuticles completely. The phenotypic rate was analysed using Fisher's exact test.

Raw_data_for-Figure6-Silencing-efficiency-of-HcHR3-or-HcβFTZ-F1-pupa.xlsx

Data brief description: In Figure 6A，larvae were injected once with dsRNA on the third day of the pupa. Samples were collected at 48 h and 72 h post-injection to evaluate the RNAi silencing efficiency of HcHR3 and HcβFTZ-F1. Pupa s injected with dsGFP at corresponding time points served as the control. Data are presented as mean ± standard error (SE) (n = 3). Quantitative real-time PCR (qRT-PCR) was performed to assess changes in HcβFTZ-F1 expression levels. Each treatment had three mechanical and three biological replicates. The ct value is the original data detected by the instrument. After calculation by the formula, 2^-∆∆Ct is obtained. We calculated the relative expression of the target gene according to the 2^-∆∆Ct. We selected EF-1a and RPL13 as internal reference genes. The expression levels are shown relative to those in dsGFP at the same point. Statistical significance of gene expression differences between the two groups was calculated using Student's t-test (ns, P > 0.05; * P < 0.05; P < 0.01; *** P < 0.001; ** P < 0.0001).

dsHcHR3-48h: Pupa in the experimental group at 48 hours after injection of dsRNA targeting the HcHR3 gene.

dsHcHR3-72h: Pupa in the experimental group at 72 hours after injection of dsHcHR3.

dsGFP-48h: Pupa in the control group at 48 hours after injection of the negative control dsGFP.

dsGFP-72h:Pupa in the control group at 72 hours after injection of dsGFP.

dsHcβFTZ-F1-48h: Pupa in the treatment group at 48 hours after injection of dsRNA targeting the HcβFTZ-F1 gene.

dsHcβFTZ-F1-72h:Pupa in the treatment group at 72 hours after injection of dsHcβFTZ-F1.