Nuclear receptor E75 regulates molting and ovarian development in Nilaparvata lugens
Abstract
E75, a member of the nuclear receptor gene family, regulates diverse biological processes, including ovarian development and fat body remodeling. The brown planthopper (Nilaparvata lugens Stål), a major rice pest, poses a significant threat to rice production. However, the role of Nilaparvata lugens E75 (NlE75) in development, particularly ovarian development and its underlying mechanisms, remains largely unknown.
Given distinct life histories of Nilaparvata lugens, we anticipated novel functions of NlE75. In this study, we found that downregulation of NlE75 expression significantly reduced the eclosion rate, especially in the third-, fourth-, and fifth-instar nymphs. Moreover, decreased NlE75 levels affected fat body morphology as well as ovarian and testicular development, leading to delayed ovarian maturation and reduced egg production. NlE75 knockdown drastically reduced the total number of eggs laid. We also found that NlE75 may participate in the transcriptional regulation of Vitellogenin (Vg), Vitellogenin-like1 (Vg-like1), and Vitellogenin-like2 (Vg-like2), and that downregulation of NlE75 significantly reduced Vg protein expression.
In summary, we found that NlE75 is required for lipid droplet formation in the fat body, as well as for ovary and vas deferens development, representing novel findings. Importantly, downregulation of NlE75 altered fat body morphology and reduced lipid content, suggesting decreased energy reserves. Furthermore, NlE75 was involved in the transcriptional regulation of cell cycle-related genes, influencing DNA replication and the expression of genes controlling cell cycle progresssion. Flow cytometry analysis revealed significant disruption of ovarian cell cycles following NlE75 downregulation, suggesting that E75 may function in cell cycle progression to coordinate development and reproduction in N. lugens. This study provides a reference for related research in other species and offers theoretical support for the future development of novel pesticides targeting genes such as NlE75.
Dataset DOI: 10.5061/dryad.g1jwstr4w
Description of the data and file structure
These are datat of the figures (and figure of SI). These are all data of qRT-PCR experiments, ecdysis and eclosion experiments, measuring of vas deferens, measurement of Vg protein(semi-quantitative), and ovarian grade analysis. These are all experiment-derived data.
Files and variables
File: Table.zip
The folder contains the data of the article.
Data of figures including Fig. 1, Fig. 2, Fig. 4, Fig. 5, Fig. 6, Fig. 7，Fig. S4 and Fig. S5.
Data of the qRT-PCR: Fig. 1A,B,C, Fig. 2ABCMN, Fig. 4A, Fig. 7.
Data of the percentage of edcdysis : Fig. 2 D,E,F,H,I,K;
Data of the percentage of emergence: Fig.2 G,J,L;
Data of the width of the vas deferens: Fig. 3I;
Data of the expression of Vg protein: Fig. 4C;
Data of the percentage of grade: Fig. 4D;
Data of the preovipositon period: Fig. 4E;
Data of the number of eggs: Fig. 4F;
Data of the percentage of lipid droplet: Fig. 5G;
Data of the length of the fat body: fig. 5H;
Data of the width of the fat body: fig. 5I;
Data of the percentage of 2N, 4N, 8N, 16N and 32N cells: Fig.6C.
Details of the data of each figure :
Table Fig. 1
Detection of NlE75 Expression Levels in Brown Planthopper Developmental Stages (Fig. 1A);
Treatment: Egg, 1st instar nymph, 2nd instar nymph, 3rd instar nymph, 4th instar nymph and 5th instar nymph, Female and Male, were collected separately for total RNA extraction, reverse transcription and qRT-PCR.
Detection of NlE75 Expression Levels in Female Brown Planthopper Tissues (Fig. 1B);
Treatment: Brain, Antenna, Proboscis, Forewing, Pronontum, Mesotergum, Leg, Foregut, Mid-gut, Malpighian, Hid-gut, Fat body, Ovarian, Lateral oviduct, Median oviduct and Ovipositor, from females, were collected separately for total RNA extraction, reverse transcription and qRT-PCR.
Detection of NlE7
5 Expression Levels in Male Brown Planthopper Tissues (Fig. 1C);
Treatment: Brain, Antenna, Proboscis, Forewing, Pronontum, Mesotergum, Leg, Foregut, Mid-gut, Malpighian, Hid-gut, Fat body, Ovarian, Lateral oviduct, Median oviduct and Ovipositor, from males, were collected separately for total RNA extraction, reverse transcription and qRT-PCR.
Table Fig. 2
Line1-8 Detection of E75 expression levels in 3rd instar nymphs after dsE75 interference (Fig. 2 A);
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Line11-18 Detection of E75 expression levels in 4th instar nymphs after dsE75 interference (Fig. 2 B);
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Line21-28 Detection of E75 expression levels in 5th instar nymphs after dsE75 interference (Fig. 2 C);
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Line31-54 Effects of dsE75 interference in 3rd instar nymphs on the phenotype of brown planthopper (Nilaparvata lugens) (including mortality in 3rd, 4th, and 5th instars, as well as emergence rate) (Fig. 2 D-G);
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Line57-71 Effects of dsE75 interference in 4th instar nymphs on the phenotype of brown planthopper (including mortality in 4th and 5th instars, as well as emergence rate) (Fig. 2 H-J);
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Line74-223 Effects of dsE75 interference in 5th instar nymphs on the phenotype of brown planthopper (including mortality in 5th instar and emergence rate) (Fig. 2 K-L);
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Line226-233 Effects of NlE75 downregulation in 5th instar nymphs on hormone-related genes (Fig. 2 M);
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Line236-252 Changes in NlE75 expression after downregulation of hormone-related genes (Fig. 2 N).
Treatment: dsGFP, dsMet, dsKr-h1, dsEcR and dsE93 injected (separately) brown planthoppers were used.
Table Fig. 3
Measurement of Male Spermiduct Width Following Downregulation of NlE75 Expression in 5th Instar Nymphs (Fig. 3 I).
Unit of measurement: μm
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Table Fig. 4
The effect of dsE75 injection on the expression levels of Vg, Vg-like1, and Vg-like2 in the head-thorax and abdomen of 5th instar nymphs (Fig. 4 A);
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Quantitative detection of Vg protein expression levels after dsE75 injection in 5th instar nymphs (Fig. 4 C);
Treatment: dsGFP and dsE75 injected (separately) females were used.
Statistical analysis of ovarian grading in emerged female adults after dsE75 injection in 5th instar nymphs (Fig. 4 D);
Treatment: dsGFP and dsE75 injected (separately) females were used.
Statistical analysis of pre-oviposition period and total egg production in emerged female adults after dsE75 injection in 5th instar nymphs (Fig. 4 E-F).
Treatment: dsGFP and dsE75 injected (separately) females were used.
Missing Values
Missing values are indicated by NA. NA stands for “Not Available”. Specific reasons are as follows:
NA: No eggs laid, therefore a preoviposition period does not exist.
Table Fig. 5
Lines 1-8: Measurement of fluorescence intensity in the fat body after injection of dsE75 into 5th instar nymphs (Fig. 5 G);
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Lines 11-41: Measurement of length and width of the fat body after injection of dsE75 into 5th instar nymphs (Fig. 5 H-I).
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Table Fig. 6
Line 1-19: Effect of dsE75 injection on DNA replication in brown planthopper ovarian cells of 5th instar nymphs, as analyzed by flow cytometry (Fig. 6 A-C). Flow cytometry was used to examine alterations in ovarian cell cycle progression after RNAi-mediated downregulation of NlE75. Ovaries were dissected three days after eclosion from dsRNA-injected N. lugens. Comparison of control samples (dsGFP) with those exhibiting reduced NlE75 expression (dsE75). Each DNA content category was analyzed. Since only Grade III and grade IV ovaries were present at this stage, these two types were selected for comparison. The analysis is based on fluorescence intensity and cell count, and the corresponding percentages are calculated according to the peak values at each stage. The instrument used is a Beckman flow cytometer (CytoFLEX S, Beckman Coulter Life Sciences, Indiana, United States), and the software used is CytExpert 2.4 (Beckman Coulter Lif Sciences, Indiana, United States).
2N, 4N, 8N, 16N, and 32N represent different ploidy levels or DNA content of a cell, relative to a baseline haploid (N) genome. These categories indicate how many copies of the haploid genome are present in a cell's nucleus. They describe either the cell's position in the cycle (2N vs 4N) or its intrinsic, stable ploidy as a polyploid cell (4N, 8N, 16N, 32N).
Table Fig. 7
Effects of injecting dsE75 into 5th instar nymphs on DNA replication and cell cycle-related gene expression (Fig. 7).
Treatment: dsGFP and dsE75 injected (separately) brown planthoppers were used.
Table Fig. S4
Lines 1-8: Detection of E75a expression levels in 5th instar nymphs after dsE75a injection (Fig. S4 A).
Treatment: dsGFP and dsE75a injected brown planthoppers were collected separately, for total RNA extraction, reverse transcription and qRT-PCR.
Lines 11-82: Effects of dsE75a injection in 5th instar nymphs on the phenotype of brown planthoppers (including 5th instar mortality and eclosion rate) (Fig. S4 B-C).
Treatment: dsGFP and dsE75a injected 5th instar nymphs were used.
Table Fig. S5
Line1-26: Detection of NlE75 expression levels in the developmental templates of the brown planthopper under different reference genes (RP11/RP15/18S) (Fig. S5 A);
Treatment: Egg, 1st instar nymph, 2nd instar nymph, 3rd instar nymph, 4th instar nymph and 5th instar nymph, Female and Male, were collected separately for total RNA extraction, reverse transcription and qRT-PCR.
Line29-78: Detection of NlE75 expression levels in the tissue templates of female brown planthoppers under different reference genes (RP11/RP15/18S) (Fig. S5 B);
Treatment: Brain, Antenna, Proboscis, Forewing, Pronontum, Mesotergum, Leg, Foregut, Mid-gut, Malpighian, Hid-gut, Fat body, Ovarian, Lateral oviduct, Median oviduct and Ovipositor, from females, were collected separately for total RNA extraction, reverse transcription and qRT-PCR.
Line81-130: Detection of NlE75 expression levels in the tissue templates of male brown planthoppers under different reference genes (RP11/RP15/18S) (Fig. S5 C).
Treatment: Brain, Antenna, Proboscis, Forewing, Pronontum, Mesotergum, Leg, Foregut, Mid-gut, Malpighian, Hid-gut, Fat body, Ovarian, Lateral oviduct, Median oviduct and Ovipositor, from males, were collected separately for total RNA extraction, reverse transcription and qRT-PCR.
Code/software
Excel.