Role of nuclear receptors NlHR3 and NlFTZ-F1 in regulating molting and reproduction in Nilaparvata lugens (stål)

The nuclear receptors HR3 and FTZ-F1 are highly conserved and function to regulate molting and reproduction in both hemimetabolous and holometabolous insects. However, their roles in Nilaparvata lugens are largely unknown. In the present study, we discover that NlHR3 and NlFTZ-F1 are activated in the nymph stages by ecdysone signaling. Transcription disruption of NlHR3 and NlFTZ-F1 expression prevents nymph ecdysis and metamorphosis, which leads to abnormal appearance, malformed ovaries, and lethal phenotypes. In addition, we demonstrate that NlHR3 and NlFTZ-F1 regulate molting and reproduction by interacting with the intrinsic 20E and JH signaling pathways. Our work offers a deep insight into the action mechanisms of HR3 and FTZ-F1 in insects. Moreover, NlHR3 and NlFTZ-F1 could properly be exploited as potential target genes for developing RNAi-based pesticides to control N. lugens.

The 20E pulse triggers a transcriptional cascade composed of 20E early-response genes, such as hormone receptor 3 (HR3) and HR4 (King-Jones and Thummel, 2005;Ruaud et al., 2010), and then induces a nuclear receptor factor, FTZ-F1, to play a developmental switch role (Dubrovsky, 2005;Zhang et al., 2021). In D. melanogaster, DmHR3 induced the expression of βFTZ-F1 by directly binding to DmHR3-binding sites and transmitted a 20E signal cascade (Parvy et al., 2014). All these results indicated that HR3 and FTZ-F1 play a crucial role in insect growth and development, and there are interactions between them and the 20E signaling pathway.
The brown planthopper, Nilaparvata lugens (Stål), is one of the most destructive pests that feed on rice plants, causing a significant threat to rice production of China and other Asian countries (Cheng et al., 2003;Heong and Hardy, 2009). Although the molecular functions of HR3 and FTZ-F1 at molting and metamorphosis stages in holometabolous insects have been well investigated, their role in N. lugens and the relationship between NlHR3, NlFTZ-F1, and the 20E signaling pathway remain unclear. In the present study, we cloned HR3 and FTZ-F1 from N. lugens and analyzed their developmental and tissue-specific expression profiles. The relationship between NlHR3, NlFTZ-F1, and the 20E signaling pathway was also investigated through RNAi, the gene expression level, and measurements of survival rates. The results will aid the elucidation of insect molting and metamorphosis mediated by nuclear receptor genes and provide potential target genes for the development of RNAi insecticides to control the brown planthopper.

Insect rearing
Brown planthoppers were reared on a susceptible rice (Oryza sativa) variety, Taichung Native 1 (TN1), cultivated at 27°C ± 0.5°C and 75% ± 5% relative humidity under a 14L: 10D (h) photoperiod, according to a previously described method . Total RNA was extracted with the TRIzol Total RNA Isolation  Kit (Invitrogen, Carlsbad, CA, United States). The concentration  and  purity  were  measured  with the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Rockford, IL, United States) and determined by agarose gel electrophoresis. cDNA was synthesized by using the ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan), following the manufacturer's manual.

Molecular cloning and sequence analysis
Based on the published N. lugens genomic and transcriptomic data (Xue et al., 2014;Wan et al., 2015), NlHR3 and NlFTZ-F1 homologies were identified and their sequences were confirmed by the reverse transcription polymerase chain reaction (RT-PCR) using primers, as shown in Supplementary Table S1. The PCR product was gel purified, ligated into the vector TOPO2.1 (Invitrogen, Carlsbad, CA), and transformed into Escherichia coli DH5α competent cells (Novagen, Darmstadt, Germany). A total of 10 recombinant plasmids from several independent subclones were fully sequenced on the Applied Biosystems 3730 automated sequencer (Foster City, CA) from both directions. The newly described transcript variants of NlHR3 and NlFTZ-F1 were submitted to GenBank.

Developmental-and tissue-specific expression profiles of NlHR3 and NlFTZ-F1
For temporal expression analysis of NlHR3 and NlFTZ-F1, total RNA samples were prepared from eggs; first (N1), second (N2), and third (N3) instar nymphs; each day of the fourth and fifth instar nymphs (N4D1, N4D2, N4D3, N5D1, N5D2, N5D3, and N5D4); and newly emerged (New-A) and 2-day-old female adults (AD2). To analyze the tissue-specific expression patterns, the different tissues were obtained by dissecting the fourth and fifth instar nymphs. The first group comprised the head (He), thorax (Th), and abdomen (Ab). The second group comprised the integument (In), wingbud (Wi), midgut (Mg), leg (Lg), and fat body (Fb). Quantitative realtime PCR (qRT-PCR) was conducted to estimate expression levels of NlHR3 and NlFTZ-F1 of various samples using internal control genes (RSP15 and RSP11), according to the published methods . All the qRT-PCR primers are shown in Supplementary Table S1. There were three independent replications of each biological sample with three technical replicates. Data were analyzed by the 2 −ΔΔCT method (Livak and Schmittgen, 2001) and in accordance with the MIQE guidelines (Bustin et al., 2009).

20E treatment
The response of NlHR3 and NlFTZ-F1 to 20E application was determined by referring to previous methods . 20E was purchased from Sigma (Sigma-Aldrich, Shanghai, China). The fourth instar nymphs (0-12 h) were collected and treated with 0.2 μL 20E (0.1 μg/μL in acetone). After treatment with 20E, samples were taken at 6, 12, and 24 h after application to extract total RNA, and the expression of NlHR3 and NlFTZ-F1 was examined. Three replicates (10*3 nymphs, a total of 30 nymphs) were used to extract total RNA, and acetone was used as the control.

dsRNA synthesis and RNAi bioassay
The dsRNA synthesis and RNAi bioassay method were applied, as previously described . The dsDNA fragments were amplified by RT-PCR using specific primers (Supplementary Table  S1) and used as templates to synthesize dsRNA using the MEGAscript T7 High-Yield Transcription Kit (Ambion, Austin, TX, United States). The quality and concentration of the dsRNA were detected and kept at −80°C until further use. The dsRNA targeting the gene-encoding green fluorescence protein (dsGFP) served as a negative control.
RNA interference (RNAi) was performed by the FemtoJet microinjector (Eppendorf), as previously reported . Approximately 200 ng and 400 ng dsRNA were microinjected into each individual of the third and fifth instar nymphs, respectively (Xu et al., 2015). A total of 250 nymphs (10 replicates) were used for each treatment, including three survival evaluation replicates, three phenotypic evaluation replicates, three qRT-PCR verification replicates, and one backup replicate. QRT-PCR verification was conducted 3 days after injection.

Data analyses
Data were analyzed using Student's t-test (the difference between two samples), Tukey's test (the difference among three or more samples), and analysis of variance (ANOVA) by data processing system software (Tang and Zhang, 2013).

Identification of the HR3 and FTZ-F1 transcripts of N. lugens
The cDNA of putative HR3 and FTZ-F1 transcripts in N. lugens was cloned and submitted to GenBank (OP937347, OP937348, KU928171.1, and KU928171.1). We identified two transcript variants of HR3 (NlHR3a and NlHR3b) that encoded 520 and 462 amino acid residues and two transcript variants of FTZ-F1 (NlFTZ-F1a and NlFTZ-F1b) that encoded 609 and 633 amino acid residues. The HR3 and FTZ-F1 family proteins contain two conserved nuclear receptor functional domains, among which the DNA-binding domain (DBD) at the amino terminal and the ligandbinding domain (LBD) at the carboxy terminal are highly conserved (Supplementary Figures S1A, S2A). The phylogenetic tree analysis showed that NlHR3 has a close relationship with that of A. pisum, and NlFTZ-F1 has a close relationship with that of A. pisum and P. humanus (Supplementary Figures S1C, S2C).

Expression profiles of NlHR3 and NlFTZ-F1
Developmental expression analysis showed that the transcript levels of NlHR3 were the highest in the first instar period ( Figure 1A), while NlFTZ-F1 had two expression peaks at the later stages of the fourth and fifth instar periods (Figure1C). The expression profiles of NlHR3 and NlFTZ-F1 in different tissues are similar and relatively higher in the thorax and appendage of the nymphs ( Figures 1B,D). The spatiotemporal data are compatible with the common idea that NlHR3 and NlFTZ-F1 play a vital role in the 20E signaling pathway.
In the nymphs injected with dsNlFTZ-F1, the transcript levels of NlCYP314A and NlHR3 were significantly decreased by 74.2% and 80.3% (p-value <0.01), respectively, and NlKr-h1 was significantly increased by 1771.3% (p-value <0.01) ( Figure 3B). In the nymphs injected with dsNlCYP314A (an ecdysone synthesis key gene), the transcript levels of NlHR3 and NlFTZ-F1 were significantly decreased by 56.6% and 64.1% (p-value <0.01), respectively ( Figure 3C), while in the nymphs injected with dsNlKr-h1 (a juvenile hormone response key gene), the transcript levels of Frontiers in Physiology frontiersin.org NlHR3 and NlFTZ-F1 were not significantly different from that of the control ( Figure 3D). Ecdysone receptor (EcR) is the starting point of an ecdysone cascade reaction and the upstream of HR3 and FTZ-F1. We analyzed the expression level of NlEcR, but there was no significant difference after the knockdown of NlHR3 and NlFTZ-F1 (Supplementary Figure S3). In summary, the transcript levels of 20E biosynthetic and cascade regulated genes were decreased significantly by silencing  NlFTZ-F1, while only the ecdysone cascade regulated genes were decreased significantly by silencing NlHR3. The transcript level of the JH response key gene was increased significantly by silencing NlHR3 and NlFTZ-F1. The transcript level of NlFTZ-F1 was decreased significantly by silencing NlHR3.

Effect of expression silencing of NlHR3
and NlFTZ-F1 on nymph-nymph molting In order to investigate the role of NlHR3 and NlFTZ-F1 in nymph-nymph molting, dsNlHR3 and dsNlFTZ-F1 from the common region (marked with black lines in Supplementary Figure S1) were synthesized in vitro and injected into the third instar nymphs. Compared to the control, the transcript levels of NlHR3 and NlFTZ-F1 were significantly decreased by 69.6% and 57.8% (p-value <0.01), respectively, 48 h after injecting with dsNlHR3 ( Figure 4D).
Upon injecting with dsRNA into the early third instar nymphs, the knockdown of NlHR3 resulted in a significantly lower survival rate (81.7%; p-value < 0.05), which started on the second day after dsRNA injection ( Figure 4D). The development of these nymphs was inhibited on the third instar stage, and all individuals (n = 60 out of 60) were unable to molt normally into the fourth instar stage and eventually died on the sixth day after dsRNA injection ( Figures 4A,D). The knockdown of NlFTZ-F1 resulted in a significantly lower survival rate (78.3%; p-value < 0.05) which started on the first day after dsRNA injection ( Figure 4D). The insects injected with dsRNA, targeting NlFTZ-F1, exhibited lethal phenotypes at approximately 3 days after injection, and the development was inhibited on the third nymph-nymph molting transition; as a result, the insect bodies became slender and extended. The insects then failed to shed their old cuticle and died ( Figure 4A). In contrast, the dsGFP-treated nymphs successfully completed all nymph-nymph molting transition, and the survival rate on the seventh day after injection was 93.3% ( Figures 4A,D). Furthermore, NlFTZ-F1 silencing caused a lower survival rate than NlHR3 from the second day after injection. The survival rate decreased to 0% on the fifth day after downregulating NlHR3, while all individuals died on the sixth day for downregulating NlFTZ-F1 ( Figure 4D).

Effect of expression silencing of NlHR3 and NlFTZ-F1 on the nymph-adult transition
In order to investigate the role of NlHR3 and NlFTZ-F1 in nymph-adult molting, dsRNAs were injected into the fifth instar Frontiers in Physiology frontiersin.org nymphs. The transcript levels of NlHR3 and NlFTZ-F1 were significantly decreased from the controls. The knockdown of NlHR3 and NlFTZ-F1 resulted in a significantly higher mortality rate (42.7% and 53.3%, p-value < 0.05) on the fourth day after dsRNA injection, and these individuals were unable to molt normally into adults; the old cuticle of the notum was split open, and the insects then failed to shed their old cuticle and died ( Figure 4A). The individuals that molt normally into female adults were dissected to observe the development of ovaries. The ovaries of female individuals injected with dsNlHR3 and dsNlFTZ-F1 were deficient of oocytes and abnormally formed. The oocytes of individuals injected with dsNlHR3 were smaller than those of the dsGFP, and the egg shell of oocytes in individuals injected with dsNlFTZ-F1 had no luster and elasticity ( Figure 4B), while the ovaries of those injected with dsGFP were full of oocytes (banana-shaped), and the egg shell of oocytes had luster and elasticity ( Figure 4B). No offspring was produced by female individuals injected with dsNlHR3 and dsNlFTZ-F1.

Discussion
In the present study, we cloned and characterized HR3 and FTZ-F1 genes in N. lugens, and their temporal and spatial expression profiles were analyzed. This is the first report of splice variants of FTZ-F1 in N. lugens. The HR3 and FTZ-F1 genes have splice variants that are widely distributed in insects, including D. melanogaster (Dubrovsky et al., 2011), A. aegypti (Li et al., 2000), Manduca sexta (Weller et al., 2001), T. castaneum (Tan and Palli, 2008), B. germanica (Cruz et al., 2008), and L. decemlineata (Liu et al., 2014). Many nuclear receptor factors are induced and temporally expressed in response to 20E (Liu et al., 2014;Sultan et al., 2014;Li et al., 2015;Zhang et al., 2022). Among them, FTZ-F1 was expressed after a pulse of 20E during the late nymph stage in almost all organs and is essential for molting and metamorphosis. In N. lugens, NlFTZ-F1 was remarkably induced before or right after each molt in the fourth and fifth instar nymph stages and was induced by 20E. This temporally specific expression was in concert with the cyclical fluctuation of the 20E titer detected in a previous study (Liu et al., 2014;Zhao et al., 2019;Zhang et al., 2022).
Our results showed that the knockdown of NlHR3 and NlFTZ-F1 in nymphs severely impaired nymph molting and metamorphosis, and RNAi nymph resulted in lethal phenotypes and abnormally formed ovaries. Ecdysones regulate molting and reproduction of N. lugens . In this study, the expression of NlHR3 and NlFTZ-F1 was significantly increased after 20E treatment (Figure 2), suggesting HR3 and FTZ-F1 were induced by 20E (Liu et al., 2014;Guo et al., 2015). NlCYP314A1 is a key gene of the 20E biosynthetic pathway and played an important role in the nymphal development (Li et al., 2017). By knocking down Survival rate of N. lugens subjected to dsNlHR3 and dsNlFTZ-F1 injection in the third instar nymphs. The SE was determined from three independent biological replicates, each with three technical replications. Different letters indicate a significant difference at a p-value < 0.05 (Student's t-test). * and ** indicate a significant difference at a p-value < 0.05 or < 0.01 (Student's t-test).
Frontiers in Physiology frontiersin.org NlCYP314A1 in N. lugens, NlHR3 and NlFTZ-F1 mRNA levels were decreased ( Figure 3C). These data demonstrated that NlHR3 and NlFTZ-F1 can participate in the growth and development regulated by 20E. Similar results have been reported in other insects. The expression pattern of DmHR3 is related to the titer of 20E in D. melanogaster, and 20E promotes the expression of DmHR3 (Carney et al., 1997). In the larvae of B. germanica, 20E directly regulates the expression of BgHR3 and ecdysis was inhibited after the knockdown of BgHR3 (Cruz et al., 2008). In L. decemlineata, LdHR3 regulated the pupation process by responding to 20E . D. melanogaster larvae died due to severe ecdysis defects, following silencing DmFTZ-F1 (Yamada et al., 2000). In B. germanica, the knockdown of BgFTZ-F1 led to growth retardation and molting failure (Cruz et al., 2008). Recently, the knockdown of HaFTZ-F1 severely damaged larval ecdysis in Helicoverpa armigera (Zhang et al., 2021), and the knockdown of HvFTZ-F1 impaired molting, pupation, and reproduction in Henosepilachna vigintioctopunctata (Liu et al., 2022). The result from this study also suggested that NlHR3 and NlFTZ-F1 could be a potential target for the RNAimediated pest control against N. lugens because of the high mortality rate after RNAi. However, the unintended effects of dsNlHR3 and dsNlFTZ-F1 on target and non-target organisms should be investigated before the commercialization of RNAi-based pest control (Pan et al., 2020;. Furthermore, the knockdown of NlFTZ-F1 resulted in varying degrees of downregulations of NlCYP314A1 (20E-induced biosynthetic genes) and NlHR3 (20E-induced cascade genes) ( Figure 3B). In D. melanogaster, the expression levels of two steroidogenic enzymes, phantom (Phm) and disembodied (Dib), were reduced in DmFTZ-F1 mutant ring gland cells (Parvy et al., 2005). BgFTZ-F1 regulates the onset of production of ecdysteroids at mid-nymphal stages in B. germanica (Cruz et al., 2008). RNAimediated knockdown of LdFTZ-F1 significantly repressed the transcription of LdPHM, LdDIB, and LdSHD, lowered the 20E titer in L. decemlineata (Liu et al., 2014). In H. armigera, the knockdown of HaFTZ-F1 lowered the intrinsic 20E titer, and reduced the expression of ecdysone receptors and 20E cascade regulated genes HaBrC and HaE75A (Zhang et al., 2021). These results indicated that the knockdown of FTZ-F1 resulted in irreversible changes to 20E biosynthetic and downstream cascade regulated genes, thereby impairing molting and metamorphosis. However, how FTZ-F1 regulates 20E biosynthetic and cascade regulated genes requires further study.
Kr-h1 is one of the crucial effectors that mediate the interactions between JH and 20E signaling pathways (He and Zhang, 2022). The expression level of NlKr-H1 was significantly increased after the knockdown of NlHR3 and NlFTZ-F1, suggesting that NlKr-H1 was negatively regulated by NlHR3 and NlFTZ-F1 (Figures 3A,B). The knockdown of NlHR3 and NlFTZ-F1 resulted in the reduction of 20E biosynthetic gene expression. These results led us to hypothesize that NlHR3 and NlFTZ-F1 could negatively regulate the JH signaling pathway by affecting 20E biosynthetic genes and the 20E titer. On the other hand, the mutual inhibition of the 20E-and JH-response pathways plays an important role in both embryonic and postembryonic phases (Uyehara et al., 2017;Liu et al., 2018;Truman, 2019;He and Zhang, 2022). Therefore, the interference of these 20E cascade regulated genes would lead to the weakening of the 20E cascade and the enhancement of the JH cascade, disrupting the normal synergy and accurate action time of the two hormone pathways and ultimately affecting the molting, metamorphosis, and reproduction processes of the brown planthopper.

Conclusion
Taken together, this is the first detailed report which shows that the nuclear transcription factors NlHR3 and NlFTZ-F1 are involved in the regulation of molting and reproduction in N. lugens. NlHR3 and NlFTZ-F1 regulate molting and reproduction by mediating the intrinsic 20E and JH signaling pathways. These results will deepen our understanding of the action mechanisms of HR3 and FTZ-F1 in insects. In addition, NlHR3 and NlFTZ-F1 could properly be exploited as potential target genes for developing RNAi-based pesticides to control N. lugens.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding authors.

SUPPLEMENTARY FIGURE S3
Effect of the knockdown of NlHR3 and NlFTZ-F1 on the expression of NlEcR. (A) Effect of knockdown of NlHR3 on the expression of NlEcR. (B) Effect of knockdown of NlFTZ-F1 on the expression of NlEcR. SE was determined from three independent biological replicates, each with three technical replications.

SUPPLEMENTARY TABLE S1
Primers used for RT-PCR, dsRNA synthesis, and qRT-PCR.