DOI: 10.5061/dryad.g1jwstr4w
title: Nuclear receptor E75 regulates molting and ovarian development in <em>Nilaparvata lugens</em>

description: 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.
description: # Nuclear receptor E75 regulates molting and ovarian development in
 *Nilaparvata lugens* Dataset DOI:
 [10.5061/dryad.g1jwstr4w](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, 1^st^ instar nymph, 2^nd^ instar nymph, 3^rd^ instar
 nymph, 4^th^ instar nymph and 5^th^ 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 5^th^ 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, 1^st^ instar nymph,
 2^nd^ instar nymph, 3^rd^ instar nymph, 4^th^ instar nymph and 5^th^
 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.