Functional Characterization of Nuclear Receptor MuFTZ-F1 in the Bean Flower Thrips, Megalurothrips usitatus

Abstract

The development of novel control strategies for the major cowpea pest Megalurothrips usitatus requires a deeper understanding of its critical molecular regulators. The nuclear receptor Fushi-tarazu factor 1 (FTZ-F1) is a conserved master regulator of insect development and reproduction, yet its function in M. usitatus remains uncharacterized. In this study, we investigated the expression and functional role of MuFTZ-F1 in this pest. RT-qPCR analysis revealed ubiquitous MuFTZ-F1 expression across all developmental stages and in major adult tissues. RNA interference (RNAi)-mediated knockdown of MuFTZ-F1 in the 2nd instar nymphs caused severe developmental defects, including impaired eclosion and significantly increased mortality. Mechanistically, silencing led to a significant reduction in the molting hormone ecdysone, accounting for the observed molting arrest. Furthermore, MuFTZ-F1 knockdown significantly decreased dopamine titers in both nymphs and female adults, suggesting its involvement in regulating this key biogenic amine beyond developmental processes. Our results provide the first functional evidence that MuFTZ-F1 is indispensable for nymphal development and survival in M. usitatus, mediated through the regulation of ecdysone. The profound lethal effect of MuFTZ-F1 silencing underscores its promise as a target for RNAi-based pest management strategies against this economically important pest.

1. Introduction

The bean flower thrips, Megalurothrips usitatus (Bagnall) (Thysanoptera: Thripidae), is a major pest of cowpea (Vigna unguiculata) worldwide, inflicting severe yield losses through direct feeding damage [1,2]. Current management practices rely heavily on conventional insecticides, but their efficacy is increasingly compromised by the evolution of resistance and growing environmental concerns [3]. This situation underscores an urgent need for novel, targeted control strategies. RNA interference (RNAi) has emerged as a promising platform, offering high specificity and potency for both functional genetic studies and next-generation pest control [4,5]. Although previous RNAi screening in M. usitatus identified several target genes (MuαCOPI, MuNICE3, MuSrp54k, and MuRop) that induced moderate mortality (23.1–34.8%) [6,7], the discovery of new, highly lethal targets remains a critical priority for developing robust biocontrol applications.

The nuclear receptor βFTZ-F1 represents a prime candidate gene. It is a conserved master regulator of insect metamorphosis and reproduction, functioning as a critical competence factor within the ecdysone signaling pathway [8]. In well-studied holometabolous insects like Drosophila melanogaster [9,10], Aedes aegypti [11], Helicoverpa armigera [12], Apis mellifera [13], Lepeophtheirus salmonis [14], and Henosepilachna vigintioctopunctata [15], βFTZ-F1 governs essential processes such as the larval-pupal transition, vitellogenesis, and ovulation. It also directly regulates ecdysteroidogenesis, establishing a feedback loop that ensures proper 20-hydroxyecdysone (20E) titers during development [12,16]. In holometabolous insects, the molting hormone 20-hydroxyecdysone (20E) initiates a hierarchical genetic cascade by binding to its heterodimeric receptor, composed of the Ecdysone Receptor (ECR) and Ultraspiracle (USP) [17]. This binding triggers the expression of early response genes, including Broad-Complex (BRC), E74, and E75. These early genes subsequently activate early-late genes, such as hormone receptor 3 (HR3) and HR4. The HR3 and HR4 proteins then feedback to repress the early genes, facilitating a critical developmental switch and inducing the expression of the nuclear receptor FTZ-F1 [18]. The expression of FTZ-F1 is essential for mediating specific physiological responses to the subsequent 20E pulse [12,15]. Furthermore, 20E signaling coordinates morphological changes such as cuticular pigmentation by regulating pigment synthesis genes, including tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC), which are involved in the sclerotization and tanning of the new cuticle [15,19]. However, despite its characterized pleiotropic roles in holometabolous species, the function of βFTZ-F1 (hereafter MuFTZ-F1) in the hemimetabolous M. usitatus remains entirely unknown. The distinct developmental pathway of thrips, which lacks a true pupal stage, raises fundamental questions about the functional conservation of this key regulator in hemimetabolous insects.

This study therefore aims to characterize MuFTZ-F1 and evaluate its potential as an RNAi target in M. usitatus. Our specific objectives are to (1) determine the spatiotemporal expression profile of MuFTZ-F1 across different developmental stages and tissues, and (2) utilize RNAi to elucidate its functional role in nymphal development and survival, including its impact on the ecdysone and dopamine signaling pathways. The findings will provide crucial molecular insights into development in a hemimetabolous pest and assess the practical viability of MuFTZ-F1 for RNAi-based management strategies.

2. Materials and Methods

2.1. Insect Rearing

A laboratory colony of M. usitatus, originally collected in 2019 from Sanya, Hainan Province, China, was maintained as previously described [20]. The insects were reared in glass jars (6 cm diameter × 10 cm height) containing fresh cowpea pods, with moistened kitchen paper at the bottom to support pupation. The colony was kept in a climate-controlled chamber at 26 ± 1 °C and 70 ± 5% relative humidity, under a 12:12 h (light–dark) photoperiod.

2.2. In Silico Analysis and Phylogenetic Analysis of MuFTZ-F1

The open reading frame (ORF) of MuFTZ-F1 was identified from our unpublished transcriptome database (see Supplementary Materials). Amino acid sequences of FTZ-F1 orthologs from 14 insect species were aligned using the MUSCLE algorithm in MEGA7 software. A Maximum Likelihood (ML) phylogenetic tree was constructed using the best-fit JTT model, selected based on the lowest Bayesian Information Criterion (BIC) score. Node support was assessed with 1000 bootstrap replicates, and values ≥ 70% were considered well-supported. The resulting tree was visualized and aesthetically optimized using the iTOL platform (https://itol.embl.de/). Trichinella pseudospiralis (Enoplida) was used as the outgroup.

2.3. Stage- and Tissue-Specific Expression Profiling of MuFTZ-F1

MuFTZ-F1 expression was analyzed across various developmental stages and tissues. For developmental stages, samples included 1st instar nymphs (50 individuals per replicate, n = 3), 2nd instar nymphs (50 individuals per replicate, n = 3), prepupae (30 individuals per, n = 3), pseudopupae (30 individuals per replicate, n = 3), and adults (20 males or females per replicate, n = 3). For tissues, midgut, ovary, salivary gland, and cuticle were dissected from about 1000 adult females per replicate (n = 3). All samples were immediately preserved in TRIzol reagent for RNA extraction and cDNA synthesis.

2.4. dsRNA Synthesis

Double-stranded RNA (dsRNA) targeting MuFTZ-F1 and a control GFP sequence was synthesized using the HiScribe™ T7 in vitro transcription kit (New England Biolabs, Ipswich, MA, USA) (Table S1). The resulting PCR products were gel-purified and used as templates for in vitro transcription (Figure S1). Synthesized dsRNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), its integrity was confirmed by gel electrophoresis, and it was stored at −80 °C until use.

2.5. Oral RNAi Bioassay

The effects of oral RNAi were assessed using a previously established feeding bioassay [7]. Briefly, fresh cowpea seeds were shelled, soaked in a 500 ng/µL dsRNA solution on ice for 30 min, and air-dried. Experimental seeds were treated with dsMuFTZ-F1, while control seeds were treated with dsGFP. Treated seeds were then placed in bioassay boxes lined with moist kitchen paper.

For the 2nd instar nymphs, one-day-old individuals were fed treated seeds for two days, with seeds replaced every 24 h. On day 3, the nymphs were transferred to fresh, untreated cowpea pods. Each replicate contained 15 nymphs, with four replicates (60 total nymphs) per treatment. Mortality and developmental progression were recorded at 12 h intervals for five days.

For female adults, two-day-old post-eclosion adults were subjected to the same two-day feeding regimen before being transferred to fresh pods.

2.6. Gene Expression Analysis Following RNAi

To assess gene silencing efficiency and its downstream effects, the 2nd instar nymphs and female adults were fed dsMuFTZ-F1 or dsGFP (500 ng/µL) for two days. Samples were collected (15 individuals per replicate, n = 3), flash-frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted using Trizol reagent (Accurate Biology, Changsha, Hunan, China) following established protocols [21]. Subsequent RT-qPCR analysis was performed to measure the transcript levels of genes in the ecdysone pathway (MuECR, MuHR3, MuHR4, MuE75) and dopamine synthesis pathway (MuDDC, MuTH) (Table S2).

2.7. Reverse Transcriptase-Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis

Gene expression was quantified by RT-qPCR using three biological and three technical replicates per gene. Primers are listed in Table S2. MuGAPDH [22] served as the internal reference gene for normalization. Relative transcript levels were calculated using the 2^−ΔΔCt method [23].

2.8. 20E and Dopamine Titer Quantification

20E titer: A standard curve was generated using serial dilutions of an ecdysteroid standard. The 2nd instar nymphs fed dsMuFTZ-F1 or dsGFP for two days were collected (210 individuals per replicate, n = 4). Samples were homogenized in ice-cold phosphate-buffered saline (PBS; 1:9, w/v), centrifuged at 5000 rpm for 10 min, and the supernatant was analyzed using an Insect Ecdysone ELISA Kit (MLBIO, Shanghai, China). Optical density (OD) was measured with a microplate reader (BIO-RAD, Hercules, CA, USA).

Dopamine titer: A standard curve was generated using serial dilutions of a dopamine standard. The 2nd instar nymphs and female adults fed dsMuFTZ-F1 or dsGFP for two days were collected (nymphs: 140 individuals/replicate, n = 4; adults: 60 individuals/replicate, n = 4). Samples were homogenized in PBS (1:9, w/v), centrifuged at 3000 rpm for 20 min, and the supernatant was analyzed using an Insect Dopamine ELISA Kit (NJJCBIO, Nanjing, China). OD was measured (iMark, BIO-RAD, Hercules, CA, USA).

2.9. Statistical Analysis

Spatiotemporal expression patterns were compared by one-way analysis of variance (ANOVA) followed by Tukey’s test; significant differences (p < 0.05) are indicated by different letters. RNAi efficiency, hormone titers, and two-group gene expression comparisons were analyzed using two-tailed unpaired t-tests. Significance levels are denoted as follows: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

3. Results

3.1. MuFTZ-F1 Cloning and Phylogenetic Analysis

The MuFTZ-F1 gene contains a 1740-bp open reading frame encoding a 579-amino acid protein. Phylogenetic analysis revealed that the FTZ-F1 proteins from M. usitatus, Thrips palmi, and Frankliniella occidentalis formed a distinct, well-supported monophyletic clade within the Thysanoptera lineage. This robust clustering confirms the close phylogenetic relationship among these thrips species and suggests a conserved evolutionary trajectory for FTZ-F1 within this order (Figure 1).

3.2. Spatiotemporal Expression Profiles of MuFTZ-F1

MuFTZ-F1 was expressed ubiquitously across all developmental stages, with significantly higher transcript levels in pseudo-pupae, followed by the 1st instar nymphs (F_5,12 = 32.953, p < 0.001). No significant differences in expression were detected among the 2nd instar nymphs, female adults, and male adults, although levels were relatively lower in female adults.

Tissue-specific expression analysis in adult females revealed the highest MuFTZ-F1 transcript levels in the cuticle, moderate expression in ovaries, and comparably low expression in salivary glands and midgut. The difference in expression between salivary glands and midgut was not statistically significant (F_3,12 = 35.869, p < 0.001) (Figure 2).

3.3. Effect of dsMuFTZ-F1 on M. usitatus Nymphs

RNAi-mediated silencing of MuFTZ-F1 was highly effective in inducing lethal developmental defects in the 2nd instar nymphs. Nymphs fed dsMuFTZ-F1 exhibited severe developmental arrest, failing to undergo ecdysis, which resulted in a significant mortality rate of 66.7%. In contrast, nymphs in the dsGFP control group developed normally (Figure 3A,B).

This mortality was linked to the disruption of key genetic and hormonal pathways. Silencing of MuFTZ-F1 led to the significant downregulation of several genes within the ecdysone signaling cascade, including MuFTZ-F1 itself (t(4) = 6.112, p = 0.004), MuHR3 (t(4) = 3.018, p = 0.039), MuHR4 (t(4) = 4.440, p = 0.011), and MuE75 (t(4) = 3.105, p = 0.036). The expression of MuVg (t(4) = 27.753, p < 0.001), a critical vitellogenin gene regulated by 20E, was also profoundly inhibited. Concurrently, transcripts encoding key enzymes for dopamine synthesis, MuTH ((t(4) = 5.033, p = 0.007) and MuDDC (t(4) = 4.011, p = 0.016), were significantly downregulated. In contrast, the expression of MuECR remained unchanged (t(4) = 1.814, p = 0.144).

At the hormonal level, dsMuFTZ-F1 treatment significantly reduced the titers of both 20E (t(6) = 4.545, p = 0.004) (Figure 3D) and dopamine (t(4) = 7.326, p = 0.002) in the 2nd instar nymphs compared to the dsGFP control (Figure 3E).

3.4. Effect of dsMuFTZ-F1 on M. usitatus Female Adults

A parallel, and often more pronounced, transcriptional suppression was observed in female adults following MuFTZ-F1 knockdown (Figure 4A). The expression of MuFTZ-F1 itself remained significantly suppressed (t(4) = 4.221, p = 0.013). This downregulation extended to other core components of the ecdysone signaling pathway, including MuECR (t(4) = 5.9720, p = 0.004), MuHR3 (t(4) = 11.338, p < 0.001), MuHR4 (t(4) = 6.840, p = 0.002), MuE75 (t(4) = −6.2812, p = 0.0033), and MuVg (t(4) = 11.384, p < 0.001). Furthermore, transcripts encoding key dopamine synthesis enzymes MuTH (t(4) = 9.943, p < 0.001), MuDDC (t(4) = 7.917, p = 0.001) were also drastically reduced.

Consequently, dsMuFTZ-F1 treatment led to a significant reduction in the dopamine titer of female adults compared to the control (t(4) = 3.950, p = 0.017) (Figure 4B).

4. Discussion

This study provides the first functional characterization of the nuclear receptor MuFTZ-F1 in the hemimetabolous pest M. usitatus. Our results confirm the conserved role of βFTZ-F1 in regulating ecdysone-dependent molting and reveal a novel, critical function in modulating dopamine signaling, with significant implications for both development and adult physiology.

Consistent with its role as a broad competence factor for ecdysone signaling, MuFTZ-F1 was ubiquitously expressed across all developmental stages and tissues. This pattern aligns with observations in diverse holometabolous insects, including Drosophila melanogaster [9], A. aegypti [11], H. armigera [12], and H. vigintioctopunctata [15]. The functional conservation of βFTZ-F1 extends to hemimetabolous lineages, as demonstrated in Blattella germanica and N. lugens [16,24]. However, the unique developmental trajectory of M. usitatus, which lacks a pupal stage, may account for the distinct timing of MuFTZ-F1 expression peaks observed here, underscoring the importance of lineage-specific investigations. Notably, its expression in the midgut suggests potential, unexplored roles in digestion or nutrient sensing.

The high expression of MuFTZ-F1 in pseudo-pupae likely reflects its role in preparing for the final nymph-adult transition, a critical molting event. While the 1st instar nymphs also showed elevated expression, we selected the 2nd instar nymphs for RNAi bioassays due to their higher RNAi sensitivity and the pronounced molting defects observable at this stage. Knockdown of MuFTZ-F1 led to a complete failure of ecdysis and high mortality, a hallmark of βFTZ-F1 disruption across insects [16,25,26]. Mechanistically, this molting arrest was directly linked to a significant reduction in ecdysone titers, consistent with findings in other species [27,28]. This supports a conserved model in which βFTZ-F1 acts upstream to regulate ecdysteroidogenesis [15]; the resulting insufficient ecdysone levels prevent the 20E pulse required to initiate the molting cascade. Thus, MuFTZ-F1 is a central regulator of the endocrine hierarchy in M. usitatus.

Beyond this conserved ecdysone link, we uncovered a novel role for MuFTZ-F1 in regulating dopamine titers. Silencing MuFTZ-F1 significantly reduced dopamine levels in both nymphs and female adults. While βFTZ-F1’s role in reproduction is well-established [13,29,30], its direct connection to dopamine regulation is a new finding. In nymphs, depleted dopamine likely exacerbates mortality by impairing post-ecdysial cuticle sclerotization (tanning), leaving individuals vulnerable even if apolysis occurs [31]. In adults, reduced dopamine may impair reproductive processes such as ovarian development or oviposition behavior, although direct evidence from fecundity assays is needed to confirm these physiological effects [32]. Future work will focus on elucidating the precise molecular mechanisms by which MuFTZ-F1 governs these hormonal pathways, including the identification of its direct transcriptional targets, such as genes involved in ecdysteroidogenesis (e.g., phantom, disembodied) and dopamine biosynthesis (e.g., tyrosine hydroxylase), and its potential crosstalk with other developmental signaling networks.

The RNAi-mediated silencing of MuFTZ-F1 in this study resulted in a significant 66.7% mortality rate in nymphs, a potency markedly higher than that achieved by targeting other genes such as MuαCOPI, MuNICE3, MuSrp54k, and MuRop (23.1–34.8% mortality) [7]. This underscores the potential of MuFTZ-F1 as a superior target for RNAi-based control. However, translating this laboratory efficacy into a robust field application requires overcoming significant hurdles, primarily dsRNA instability. Our recent work identified MudsRNase3 as a key barrier in M. usitatus [6], demonstrating that its knockdown enhances RNAi efficacy and that the purified enzyme efficiently degrades dsRNA in vitro.

To address this challenge, advanced formulation strategies are critical. Nanocarriers such as chitosan, star polycations (SPc), and layered double hydroxides (LDH) have been shown to protect dsRNA from degradation by gut nucleases and UV radiation, improve cellular uptake, and enhance RNAi efficacy in thysanopterans [33,34,35]. For example, SPc nanoparticles significantly boosted dsRNA stability and RNAi efficiency in T. palmi [33]. Similarly, chemical modifications like phosphorothioate and 2′-fluoro substitutions can increase dsRNA nuclease resistance, as demonstrated in D. melanogaster cell cultures [36]. Therefore, a primary future direction is to evaluate the efficacy of chemically modified dsMuFTZ-F1 and nanoparticle-encapsulated dsRNA for controlling M. usitatus.

A central consideration for deploying dsRNA in pest management is the potential for unintended effects on non-target organisms. Current evidence suggests that the activity spectrum of dsRNA is typically narrow, with species closely related to the target pest being the most susceptible [5]. For instance, dsRNAs targeting genes in the pest H. vigintioctopunctata (dsHvHel25E and dsHvSpr54k) showed no adverse effects on the predatory ladybeetle Propylaea japonica, even at concentrations ten times higher than the lethal dose for the target pest [37]. Conversely, some studies have documented off-target effects; dsRNA targeting vATPase A from the western corn rootworm, Diabrotica virgifera virgifera negatively impacted several ladybeetle species under worst-case exposure scenarios [21].

To pre-emptively assess the risk associated with dsMuFTZ-F1, we employed the web-based tool dsRNAEngineer [38] to evaluate its potential for off-target effects for four representative predator species: Chrysoperla carnea, Galendromus occidentalis, Harmonia axyridis, and P. japonica. The analysis predicted a low probability of off-target silencing in these species (Figure S2). However, the ultimate risk is influenced by multiple factors, including the intrinsic RNAi sensitivity of non-target organisms, environmental dsRNA concentration and stability, and the position of sequence mismatches within the siRNA region. Therefore, while initial computational and laboratory data are promising, a comprehensive and species-specific risk assessment of dsMuFTZ-F1 remains a necessary prerequisite before its field application for the control of M. usitatus.

5. Conclusions

In conclusion, this study identifies the nuclear receptor MuFTZ-F1 as a critical developmental regulator in M. usitatus. Its indispensability for nymphal survival is mediated through the control of ecdysone titers required for molting, while a newly discovered role in dopamine regulation indicates additional functions in cuticle sclerotization and adult reproduction. The potent lethality resulting from RNAi silencing, which simultaneously disrupts these two pathways, establishes as a high-value target for developing RNAi-based biopesticides against this major cowpea pest.