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. 2025 Sep 24;8:1355. doi: 10.1038/s42003-025-08724-2

A case study on the γ-octalactone induced expression of Obp83g-2 in Bactrocera dorsalis (Hendel) revealed the transcriptional regulation of insect odorant binding protein

Xiaofeng Chen 1,2, Quan Lei 1, Changhao Liang 1, JinJun Wang 1,✉, Hongbo Jiang 1,✉
PMCID: PMC12460821  PMID: 40993198

Abstract

As crucial components of the insect olfactory system, odorant binding proteins (OBPs) are involved in detecting environmental chemical cues. Expression alterations of OBPs induced by odorants are conserved in many species. It presents an intriguing initial screening tool when searching for novel OBP-odorant interaction. However, the transcriptional regulation mechanism that causes this expression alteration of OBPs still remains unclear. Here, we reported a case study on the transcriptional regulation of OBP in an invasive species, Bactrocera dorsalis, upon γ-octalactone (a host volatile that strongly attracts its females to lay eggs) induction. We identified OBP83g-2 as a key OBP was involved in γ-octalactone perception through in vitro and in vivo functional assay. In addition, we found transcription factor ADF-1-like positively regulated the expression of Obp83g-2 upon γ-octalactone induction through expression pattern analysis, dual-luciferase reporter system, electrophoretic mobility shift assay (EMSA) and RNAi. Based on this, we proposed a model for the transcriptional regulatory mechanism of OBP gene in B. dorsalis. Our data not only highlights the significant role of OBP83g-2 in γ-octalactone mediated oviposition behavior, but also provides a theoretical foundation for a deeper understanding of the transcriptional regulation of OBPs triggered by external odorants in insects.

Subject terms: Entomology, Behavioural ecology

A case study on the transcriptional regulation of an odorant binding protein in the invasive fruit fly Bactrocera dorsalis demonstrates that ADF-1-like positively regulates expression of Obp83g-2 in response to the host fruit-released volatile γ-octalactone.

Introduction

Insects rely on the olfactory proteins within their sophisticated olfactory system to perceive chemical cues in the environment for seeking a host, mating and selecting an oviposition site. Odorant binding proteins (OBPs) represent the first step of this chemical perception process1. For example, NlugOBP8 of Nilaparvata lugens is involved in recognizing two rice plant volatiles linalool and caryophyllene oxide2. SfurOBP11, NlugOBP8, and LstrOBP2 of three notorious rice pests (Sogatella furcifera, N. lugens, and Laodelphax striatellus) play important roles in locating rice plants3. More and more evidences show OBPs play a crucial role in identifying and binding chemical cues that guide insect oviposition behavior. In Holotrichia parallela, HparOBP3 is found to detect volatiles derived from organic fertilizers, influencing female oviposition behavior4. Similarly, in two mosquito species, CquiOBP1 of Culex quinquefasciatus, and OBP56d-like of Aedes albopictus are found to guide oviposition site selection5,6. Whiteflies utilize OBP1 and OBP4 to perceive β-ionone in the selection of the suitable egg-laying sites on host plants7.

It has been well documented that exposure to odorants leads to reliable alterations in the mRNA levels of OBPs in various animals8,9. For instance, the expression levels of seven OBPs are significantly upregulated by sex pheromones induction in Anthonomus grandis8. In Queensland fruit fly, three OBPs show significant up-regulation in response to zingerone10. When Holotrichia oblita is exposed to (E)-2-hexenol and phenylethanol, the expression levels of HoblObp13 and HoblObp9 are rapidly upregulated11. It has been widely utilized to construct high-throughput screening to identify potential OBP–odorant interactions11–13. However, the transcriptional regulation mechanism that causes the expression alteration of OBPs still remains unclear.

Transcription factors (TFs) are key factors regulating the expression of genes. TFs play a crucial role in olfactory perception by regulating the development of olfactory sense organs, the maturation of olfactory receptor neurons, and the expression of ORs in the model insect, Drosophila melanogaster14–18. Recently, two case studies have shown the regulatory relationship between TFs and OBPs in insects. BarH1 regulates the expression of Obp19 directly and influences camphene reception in Monochamus alternatus Hope19. In another study, Dll regulates the expression of NlObp8 and NlCsp10 upon linalool induction in N. lugens20.

The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive and invasive agricultural pests. This fly poses a significant threat to over 600 fruit and vegetable crops21. A single female can lay over 3000 eggs in her whole life under optimal condition22. Many volatile components served as the chemical cues for the fly to lay eggs. For example, low concentration of β-caryophyllene attracts B. dorsalis to lay eggs23. A volatile component, 3-hexenyl acetate, that is produced by bacterium in host fruits attracts B. dorsalis females to lay eggs24. Several volatiles released by host mango, including γ-octalactone, 1-octen-3-ol, ethyl tiglate and benzothiazole, instigate laying eggs in gravid B. dorsalis females25,26. Notably, γ-octalactone has a strong attraction to guide oviposition behavior of females in various fruit fly species, including B. dorsalis and Bactrocera tryoni27. Unfortunately, it is unknown which OBPs are responsible for γ-octalactone perception so far. Our previous comparative transcriptomic study reveals that two OBPs are upregulated by γ-octalactone induction. In addition, the mRNA levels of two TFs are also upregulated28. To identify the OBPs involved in γ-octalactone perception and to elucidate the underlying transcriptional regulation mechanism, we conducted the study as follows.

In this study, we identified OBP83g-2 as a key OBP involved in γ-octalactone perception through functional genomics technology. Moreover, we found a TF, ADF-1-like, which was upregulated by γ-octalactone induction. Then we further discovered it regulated the expression of Obp83g-2 directly. Taken together, we proposed a model for the regulatory mechanism of the transcriptional expression of the OBP gene in B. dorsalis. Our study highlights the significant role of OBP83g-2 in mediating the oviposition behavior induced by γ-octalactone in B. dorsalis. Moreover, it provides a theoretical foundation for a deeper understanding of the cascade of responses triggered by external odorants in insects.

Results

Expression patterns of OBPs and TFs by γ-octalactone induction

Based on the previous γ-octalactone induced transcriptome28, we analyzed the expression levels of Obp83g-2 and Obp50e by RT-qPCR (Fig. 1A). The expression levels of Obp83g-2 and Obp50e were significantly upregulated 2.8-fold and 1.2-fold under γ-octalactone exposure, respectively. In addition, the previous study also shows that the expression levels of two TFs, Wrky-like and Adf-1-like, are upregulated28. We also analyzed the expression levels of two TFs by RT-qPCR. The Adf-1-like expression increased 2.1-fold. But the expression of Wrky-like remained steady by γ-octalactone induction (Fig. 1B).

Fig. 1. Expression patterns of OBPs and TFs by γ-octalactone induction.

Fig. 1

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A Expression patterns of two OBP genes by γ-octalactone induction. Data are means ± SE (n = 3). Asterisks represent a significant difference determined by an independent t-test (*<0.05, **<0.01). B Expression patterns of two TF genes after γ-octalactone induction. Data are means ± SEM (n = 3). Asterisks represent a significant difference determined by an independent t-test (*<0.05).

OBP83g-2 exhibited strong binding to γ-octalactone in vitro

OBP83g-2 and OBP50e were expressed and purified by a prokaryotic expression system. The recombinant proteins were confirmed by both SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. Expected molecular weights of OBP83g-2 and OBP50e were 17 kDa and 26 kDa, respectively. The SDS-PAGE results showed OBP83g-2 and OBP50e were observed with discernible bands between 15–25 kDa and 25–35 kDa, respectively (Fig. 2A, B). Then OBP83g-2 and OBP50e were further confirmed by western blot. OBP83g-2 and OBP50e presented a specific band between 15–25 kDa and 25–35 kDa, respectively. The pET30a(+) vector as control presented no band (Fig. 2A, B).

Fig. 2. Binding affinity of OBPs to γ-octalactone.

Fig. 2

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A SDS-PAGE and western blot analysis of recombinant protein OBP83g-2, Lane M: protein standards, Lane a: purified protein OBP83g-2, Lane b: total proteins from pET30a (+). B SDS-PAGE and western blot analysis of recombinant protein OBP50e, Lane M′: protein standards, Lane a′: purified protein OBP50e, Lane b′: total proteins from pET30a (+). C Binding affinity of OBP83g-2 for γ-octalactone. D Binding affinity of OBP50e for γ-octalactone. Data are means ± SEM (n = 3). MST fit curves are constructed using NanoTemper analysis software v2.2.4.

Microscale thermophoresis (MST) was performed to test the binding affinity between OBPs and odorants. OBP83g-2 strongly bound to γ-octalactone with a binding affinity value Kd of 0.26 ± 0.16 μM (Fig. 2C). Moreover, it also showed strong binding affinity to 1-octen-3-ol (Kd = 26.65 ± 6.02 μM). However, it had weak binding affinity to ethyl tiglate (Kd = 146 ± 24 μM), hexyl alcohol (Kd = 164 ± 64 μM), and benzothiazole (Kd = 1350 ± 233 μM) (Supplementary Fig. 1). OBP50e bound to three odorants in a dose-dependent manner. OBP50e showed weak binding affinity to γ-octalactone (Kd = 83.78 ± 11.63 μM) (Fig. 2D) and ethyl tiglate (Kd = 0.96 ± 0.24 mM) (Supplementary Fig. 1). Moreover, it had strong binding to (±)-Citronellal (Kd = 23.78 ± 6.63 μM) (Supplementary Fig. 1).

The sensitivity of Obp83g-2−/− mutant to γ-octalactone significantly decreased

A gRNA-directed CRISPR–Cas9 system was employed to knock out Obp83g-2. Two target sites were designed in exon 1 (Supplementary Fig. 2A). We then injected the mixture of gRNA and Cas9 into 94 freshly eggs. Six adults were obtained after embryo injection. Mosaic generation 0 (G0) individuals were identified based on the analysis of agarose gel electrophoresis and Sanger sequencing. And the G0 mutation efficiency was 16%. The clone Sanger sequencing data further confirmed a 52-bp deletion occurred in the mutation, causing a translation frameshift and early terminating of translation (Fig. 3A). The 52-bp deletion mutant strain was cultured for further analysis.

Fig. 3. Mutagenesis of Obp83g-2 and phenotypic assay.

Fig. 3

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A Sequence analysis of the mutation type and wild type (WT). The sequence alignment between the WT and the long fragment deletion mutant is shown at the top. Dots are used to represent the omitted bases. The gRNA sequences are marked with underscores. The sequence alignment between the WT and other short fragment mutants is shown at the down. Gray letters represent deleted base pairs, red letters represent mutated base pairs, and small blue letters represent inserted base pairs. B EAG responses of WT and Obp83g-2−/− mutant to different concentrations of γ-octalactone. Data are means ± SEM (n = 4). Asterisks represent a significant difference determined by an independent t-test (*<0.05, ** <0.01). C Oviposition behavior after γ-octalactone induction. Data are means ± SEM (n = 4). Asterisks represent a significant difference determined by an independent t-test (***<0.001).

After obtaining the Obp83g-2−/− mutants, we performed phenotypic analysis. We compared the Electroantennogram (EAG) response to γ-octalactone between wild type (WT) and Obp83g-2−/− mutant flies. The average EAG response of WT and Obp83g-2−/− flies both increased gradually with the concentration of γ-octalactone (Fig. 3B), reaching a peak at 10% (v/v) γ-octalactone. The EAG responses of Obp83g-2−/− flies were significantly lower at 1 and 10% (v/v) γ-octalactone compared to the WT flies. However, their EAG response showed no difference at 0.1% (v/v) γ-octalactone. Next, we did behavioral analysis between WT and Obp83g-2−/−gravid females by γ-octalactone induction (Fig. 3C). The results revealed both the WT and Obp83g-2−/− flies were attracted by γ-octalactone. But the landing frequency of Obp83g-2−/− flies was significantly decreased by 72%. The oviposition puncture frequency of Obp83g-2−/− flies was significantly decreased by 71%. Furthermore, the number of eggs laid by Obp83g-2−/− mutant females was significantly reduced by 68%. The oviposition behavior of the WT and Obp83g-2−/− flies are shown in more detail in Supplementary Movies 1 and 2.

The activity assay of the 5′-flanking promoter of Obp83g-2

In order to explore how Obp83g-2 expression is regulated, we analyzed the activity of the Obp83g-2 promoter first. The promoter sequence of Obp83g-2, comprising 967 bp upstream of the ATG site, was cloned and engineered into several promoter truncations. The luciferase activity was measured 48 h after transfection of the recombinant plasmid into Sf9 cells. The –967 to –1 bp fragment showed significantly higher activity compared to the –667 to –1 bp fragment. The activity was significantly increased 4.5-fold, when the –598 to –1 bp fragment was truncated from the –667 to –1 bp fragment. As the promoter fragment was further truncated, the activity gradually decreased between –598 and –449 bp. Moreover, –449 to –1 bp and –151 to –1 bp promoter fragments showed similar activity levels, which were reduced compared to the –508 to –1 bp promoter fragment (Fig. 4A).

Fig. 4. ADF-1-like regulated promoter activity through directly binding to the Obp83g-2 promoter.

Fig. 4

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A Basic activity analysis of the promoter of Obp83g-2. Data are means ± SEM (n = 3). Different letters above the bars indicate significant differences based on ANOVA followed by post-hoc Tukey’s HSD (p <0.05). B Effect of TFs on expression driven by the promoter of Obp83g-2. Data are means ± SEM (n = 3). Different letters above the bars indicate significant differences based on ANOVA followed by post-hoc Tukey’s HSD (p <0.05). C Electrophoretic mobility shift assay (EMSA) of ADF-1-like protein to the biotin-labeled probe of the Obp83g-2 promoter part.

The transcription factor ADF-1-like regulated the expression of Obp83g-2

Based on RT-qPCR results, Adf-1-like might play an important role in γ-octalactone perception. To further clarify the regulatory relationship between ADF-1-like and Obp83g-2, the 967-bp fragment upstream of the translation start site (ATG) of Obp83g-2 was selected for TF binding site prediction. The result showed that one ADF-1 binding site was found at –598 to –449 bp (Supplementary Fig. 3). Then, we further analyzed the expression patterns of Obp83g-2 and Adf-1-like. The expression patterns of Obp83g-2 and Adf-1-like were similar in different tissues. Both of Obp83g-2 and Adf-1-like were highly expressed in head cuticles and showed gender difference (Supplementary Fig. 4A). At different development stages, both of Adf-1-like and Obp83g-2 were highly expressed in adult. The expression levels of Adf-1-like and Obp83g-2 increased significantly after 7 d (Supplementary Fig. 4B, C).

The CDS sequence of Adf-1-like was cloned into the pIB/V5 expression vector and cotransfected into Sf9 cells with pGL3 − 967/–1. Wrky-like also were cloned into the pIB/V5 expression vector. The expression of ADF-1-like increased the activity of the Obp83g-2 promoter (Fig. 4B). The fluorescence activity was improved 3-fold after expressing ADF-1-like. The fluorescence activity of the promoter did not change significantly after expressing WRKY-like and pIB/V5 vector. To test whether ADF-1-like directly binds to the Obp83g-2 promoter, an electrophoretic mobility shift assay (EMSA) was performed. The recombinant His-ADF-1-like protein was obtained in the supernatant of Escherichia coli BL21 (DE3) cells and used for EMSA assay. When the recombinant His-ADF-1-like protein was incubated with the biotin-labeled probe, a distinct shifted band appeared (Fig. 4C). This band became weaker when the supernatant was incubated with 10-fold and 20-fold excess of unlabeled probe, and completely disappeared with a 50-fold excess of unlabeled probe. When His-ADF-1-like protein was incubated with the biotin-labeled mutant probe, there was no shifted band. These results indicate that ADF-1-like could bind to the promoter probe specifically, and this binding could be depressed with higher concentrations of unlabeled probe.

RNAi-mediated knockdown of Adf-1-like decreased the sensitivity of B. dorsalis to γ-octalactone

The expression of Adf-1-like was knocked down using RNAi. The result showed the expression level of Obp83g-2 was significantly downregulated following the knockdown of Adf-1-like (Fig. 5A). The average EAG responses of the dsAdf-1-like-treated group decreased by 54% compared to the dsGFP-treated group by 10% (v/v) γ-octalactone induction. However, there were no differences at the other two lower concentrations (Fig. 5B). Moreover, the oviposition behavior assay showed that the sensitivity of flies to γ-octalactone was significantly decreased after RNAi. The landing frequency and puncture frequency of females was significantly decreased by 52%, 51%, respectively. In addition, the number of eggs laid by females was significantly reduced by 62% (Fig. 5C).

Fig. 5. Effect of Adf-1-like knockdown on the γ-octalactone perception of B. dorsalis.

Fig. 5

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A The expression of Obp83g-2 and Adf-1-like after RNAi. Data are means ± SEM (n = 3). Asterisks represent a significant difference determined by an independent t-test (*<0.05). B EAG responses of dsGFP and dsAdf-1-like to different concentrations of γ-octalactone. Data are means ± SEM (n = 4). Asterisks represent a significant difference determined by an independent t-test (*<0.05). C Oviposition behavior after induced by γ-octalactone. Data are means ± SEM (n = 4). Asterisks represent a significant difference determined by an independent t-test (*<0.05, ** <0.01, ***<0.001).

Discussion

Numerous studies have found that OBPs exhibit odorant-induced expression alteration11,29,30. It has been successfully conducted a high-throughput screen to identify potential OBP–odorant interactions11–13. However, there are still mismatches between expression alterations and functional assays. For instance, in the scarab beetle H. oblita, the expression levels of HoblObp13, HoblObp9 and HoblObp4 are significantly upregulated by (E)-2-hexenol induction. Nonetheless, comprehensive functional analysis reveals that only HoblOBP13 and HoblOBP9 play crucial roles in (E)-2-hexenol perception11. In Glossina f. fuscipes, the expression of GffObp19a, GffObp83a1, GffObp83a4, and GffObp99d is significantly induced by 1-octen-3-ol. However, the subsequent results show GffObp19a has less effect on the perception of 1-octen-3-ol12. In our case, the mRNA levels of two OBPs responded to γ-octalactone induction, while only OBP83g-2 exhibited a strong binding affinity to γ-octalactone. It indicated unspecific interactions occured, causing the observed “false positive” alterations in gene mRNA levels11,12.

In the affinity assay, we found OBP83g-2 bound γ-octalactone and 1-octen-3-ol with Kd value of 0.26 ± 0.16 μM and 26.65 ± 6.02 μM, respectively. OBP50e bound (±)-Citronellal with Kd value of 23.78 ± 6.63 μM. These OBPs showed strong binding affinity with γ-octalactone, 1-octen-3-ol and (±)-Citronellal. Similar results have been found in other studies. For example, SzeaOBP1 showed the strongest affinity for cetanol (2.748 ± 0.139 μM) and the weakest affinity for myrcene (30.27 ± 0.798 μM)31. BminOBP9 bound strongly β-caryophyllene (1.61 μM), and BdorGOBP99a bound strongly to limonene (4.06 μM) and ocimene (4.13 μM)32. Based on binding affinity results, we knocked out Obp83g-2 through CRISPR/Cas9-mediated gene editing. The role of γ-octalactone in the oviposition behavior of B. dorsalis has been clarified in previous studies25–27. Before conducting the Obp83g-2−/− mutants behavior assays, we analyzed the effects of γ-octalactone to WT flies and obtained the similar results with previous studies (Supplementary Fig. 5). By comparing the behavior differences between the two groups, the sensitivity of the Obp83g-2−/− mutants to γ-octalactone was significantly decreased while not completely abolished. Similar results have been reported in other species, such as Spodoptera frugiperda, Chilo suppressalis, and Conogethes punctiferalis33–36. It might be associated with the involvement of multiple OBPs in the perception of the same odorant35. A previous study reported the similar phenomenon that B. dorsalis males can still perceive methyl eugenol after knocking out Obp69a37. Upon the loss of one functional OBP by gene editing, the other relative OBPs will compensate to function normally.

It was discovered in many species that odorants exposure leads to reliable alterations in mRNA levels of OBPs. However, only a single case reported that the TF regulates the alternative expression of OBPs by odorants induction. In N. lugens, the mRNA levels of NlObp8, NlCp10 and TF Dll change under linalool exposure. More results indicated Dll positively regulates the transcription of NlObp8 and NlCp10 directly20. In our previous transcriptome results, the expression level of two TFs (Adf-1-like and Wrky-like) corresponding to γ-octalactone induction28. Here, we found the expression of Wrky-like was inconsistent between RNA-seq and RT-qPCR results. A similar result occurs in many studies38. The upregulated expression of Wrky-like in RNA-seq might be a false positive. Combining the results of RT-qPCR, dual-luciferase system, TFs binding site prediction and EMSA, we speculated that ADF-1-like was a potential TF that regulated the expression of Obp83g-2.

Furthermore, we knocked out Adf-1-like using the CRISPR/Cas9 system. Unfortunately, no mutations were observed in all emerged adults of G0, as indicated in Supplementary Fig. 2B. ADF-1-like has the same conserved domain as ADF-1 of the MYB family39. Several studies have indicated that ADF-1 plays a role in embryonic development and memory formation40,41. Thus, we speculated that knocking out Adf-1-like is lethal to this fly. Alternatively, RNAi was employed to knock down the transcriptional expression of Adf-1-like. The sensitivity of gravid females to γ-octalactone was significantly decreased after RNAi treatment. It strongly supported that ADF-1-like regulate the expression of Obp83g-2. The odorant receptor co-receptor is an obligatory odorant receptor and indispensable in odor perception. In our previous studies, we cultured BdorOrco−/− mutant flies by CRISPR syetem42. We further analyzed the expression level of Obp83g-2 in BdorOrco−/− mutant by γ-octalactone induction (Supplementary Fig. 6). The results showed that the expression level of Obp83g-2 was not altered by γ-octalactone induction. It further indicated that ADF-1-like regulated the expression of Obp83g-2 under γ-octalactone induction.

Taken together, we proposed a model for the molecular regulatory mechanism of γ-octalactone perception (Fig. 6). When γ-octalactone enters the olfactory sense organs, OBP83g-2 identifies and transports it to the olfactory neurons membrane, where γ-octalactone binds to ORs. Activation of ORs triggers the opening of cyclic nucleotide-gated, ion channels, resulting in membrane depolarization, and the generation of action potentials in olfactory sensory neurons43. This electrical signal is transmitted to the central nervous system to guide insect behavior. Meanwhile, ORs activation increases intracellular calcium (Ca2+) and cAMP levels, activating downstream signaling pathways44,45. These pathways ultimately lead to the activation of ADF-1-like, which initiates transcription of Obp83g-2 by γ-octalactone induction. However, the specific details of this signaling cascade remain poorly understood and warrant further investigation to elucidate the underlying mechanisms.

Fig. 6.

Fig. 6

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Schematic diagram of the transcriptional regulation mechanism of Obp83g-2.

Maintaining a highly sensitive olfactory system incurs both metabolic and ecological costs. Thus, insect olfaction is typically plastic and responds behaviorally to chemical stimuli only in specific contexts. It is an effective strategies to balance the highly sensitive olfactory system and metabolic through this induction of transcriptional regulation. Our finding provides a theoretical foundation for a deeper understanding of the cascade of responses triggered by external odorants in insects. Moreover, it’s helpful to reveal the regulatory strategies.

Conclusions

Our study highlights the significant role of OBP83g-2 in mediating the oviposition behavior of B. dorsalis induced by γ-octalactone. Moreover, we proposed a model for the regulatory mechanism of the transcriptional expression of the OBP gene in insects for the first time. Our research provides a theoretical foundation for a deeper understanding of the cascade of responses triggered by external odorants in insects, aiding in the interpretation of the overall physiological process.

Methods

Insects

The WT B. dorsalis was collected from Haikou, Hainan province, China, in 2008. All of the B. dorsalis strains were maintained at 27 °C, 70–5% RH, and a 14:10 h (L:D) photoperiod in the laboratory. All of the larvae and adults were maintained in metal cages with artificial feed. The artificial diet for adults consisted of honey, sugar, yeast powder, and vitamin C. The artificial diet for larvae consisted of corn, wheat germ flour, yeast powder, agar, sugar, sorbic acid, vitamin C, linoleic acid, and filter paper.

RT-qPCR

15-day-old (15 d) gravid females were induced by 10% (v/v) γ-octalactone (Sigma-Aldrich, St. Louis, MO, USA), which was diluted in mineral oil (Sigma-Aldrich). After 2 h, the female heads were collected as treatment group samples. The control group was treated with mineral oil. Each group had three independent biological replications, and each replication contained heads from 10 individuals. The 15 d females and males were dissected to collect different tissue samples, including antennae (An), head cuticles (Head), proboscis (MO), maxillary palps (MP), legs (Leg), wings (Wing) and ovipositors (OV). Each group had three independent biological replications and each replication contained 15–30 individuals. Then flies at different developmental stages were also collected, including egg, larva, pupa and adult. In addition, the heads of flies at different adult stages (1 d, 3 d, 7 d, and 15 d) were also collected. Each group had three independent biological replications and each replication contained 4–6 individuals.

Total RNA was extracted using the standard TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA) protocol. RQI DNase (Promega, Madison, WI, USA) was used to remove any genomic DNA contamination from the RNA samples. The cDNA was synthesized using PrimeScript RT reagent kit (TaKaRa, Dalian, China) and stored at –20 °C. RT-qPCR was performed using SYBR Supermix (Novoprotein, Shanghai, China) following the manufacturer’s instructions in a 96-well plate with the CFX Connect™ Real-Time System. All the primers were designed with the online website Primer3 (http://primer3.ut.ee/) (Supplementary Table 1). The double internal reference genes α-tubulin (GenBank: GU269902) and rps3 (GenBank: XM_011212815) were applied because of their stable expression level in B. dorsalis. The relative expression level was calculated using the 2–∆∆Ct method46.

Protein expression and binding affinity assay

OBP83g-2 and OBP50e were expressed using a prokaryotic expression system. The coding sequences were amplified by PCR without the signal peptide. The primers were shown in Supplementary Table 1. Purified PCR product was transferred to the expression vector pET-30a (Thermo Fisher Scientific). The recombinant plasmid was then transformed into E. coli BL21 (DE3) (Tiangen, Beijing, China) for protein production. The recombinant protein was induced by isopropyl-β-d-thiogalactopyranoside. The recombinant protein was purified by Ni-NTA affinity chromatography (Qiagen, Hilden, Germany). SDS-PAGE confirmed that the recombinant protein was soluble. His-tag antibody (1:1000) (Beyotime, Beijing, China) and HRP-conjugated secondary antibody (1:5000) (Beyotime) were used to perform a western blot to further confirm the size of the recombinant protein. The concentration was determined by a BCA protein assay kit (Beyotime).

We determined the binding affinity between OBPs and odorants by MST. The protein with His-tag was labeled using the Monolith Protein His-Tag Labeling Kit RED Tris-NTA 2nd Generation (Nano Temper Technologies, Munich, Germany) according to the manufacturer’s instructions. The odorants (γ-octalactone, 1-octen-3-ol (Sigma-Aldrich), ethyl tiglate (Sigma-Aldrich), (±)-Citronellal (Sigma-Aldrich) were dissolved in 99.99% DMSO (Sigma-Aldrich) and prepared as 16 serial dilutions (1:1) in phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST) and 0.2% DMSO. The mixture of protein and odorant was loaded into Monolith NT.115 Standard Treated Capillaries (NanoTemper Technologies) and tested on a Monolith NT.115 instrument (NanoTemper Technologies) at 25 °C. The samples were loaded and measured at 60% LED/excitation power and at medium MST power. Three independent replicates were analyzed to evaluate the binding affinity (Kd) using NanoTemper analysis software (version 2.3.0). We take the Kd value below 30 μM as strong binding affinity13.

CRISPR/Cas9 mediated mutagenisis

The exon sequence of Obp83g-2 was predicted using the high-quality B. dorsalis genome assembly. Each gRNA sequence was 20 nucleotides in length plus NGG as the protospacer adjacent motif (Supplementary Table 1). The gRNA was synthesized and purified using the GeneArt Precision gRNA Synthesis Kit and GeneArt gRNA Clean-up Kit (Thermo Fisher Scientific). The purified gRNA (final concentrations: 600 ng/μL) and Cas9 protein (Thermo Fisher Scientific) (final concentrations: 500 ng/μL) were mixed for injection. The embryo injection was carried out following the previously described13.

The virgin G0 survivors were individually crossed to WT flies (single pair) to collect G1 offspring. The genomic DNA of the G0 adults was extracted using the TIANamp Genomic DNA Kit (Tiangen) after collecting enough G1 eggs. Then, genomic DNA of offspring was extracted from one leg using InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA, USA). The genomic DNA was used as the template for PCR amplification. The PCR products were distinguished by the agarose gel electrophoresis banding patterns and Sanger sequencing. Heterozygous G1 flies were backcrossed with WT flies for at least five generations to ensure stable transmission of the mutation. Then the homozygous mutant flies were obtained by heterozygotes inbreeding.

Phenotypic analysis of Obp83g-2–/– mutants

EAG assays: The antenna was fixed to two electrodes with electrode gel Spectra 360 (Parker, Fairfield, NJ, USA). γ-octalactone was diluted to 10, 1, and 0.1% (v/v) with mineral oil to serve as the electrophysiological stimulus. And mineral oil was used as a negative control. Odorant stimulation lasted 1 s, and the interval between two stimulations was at least 60 s. The constant air flow (200 ml/min) was produced by a controller (Syntech, Hilversum, Netherlands) to stimulate the antenna. EAG responses were recorded using EAG-2000 software (Syntech) with an IDAC4 amplifier (Syntech). Both 10–15 d WT and OBP83g-2–/– mutants were detected at least 15 individuals. Each group had four independent biological replications.

Behavior assay: oviposition behavior of flies was monitored in a 12 × 12 × 12 cm transparent cage. A Petri dish (diameter: 3 cm) filled with 1% agar was served as an oviposition substrate, and 20 μL 10% (v/v) γ-octalactone were added at the central of the dish. The agar disc was covered in a pierced plastic wrap to mimic fruit skin, encouraging flies to extend their ovipositor into the plastic wrap to lay eggs and was placed at the center of the cage. Five females were placed in a transparent cage to acclimate to the environment for 3–4 h before the experiment. A Sony FDR-AX40 camera was placed in front of the cage to record the behavior of the flies for 24 h. The first 3 h duration (1–3 h) of the video was selected to analyze the landing frequency and ovipositor puncture frequency. The number of eggs was counted after 24 h. Each group had four independent biological replications.

Cloning of the Obp83g-2 5′-flanking regions and luciferase reporter assays

Based on B. dorsalis genomic DNA, the fragment comprising 967 bp upstream of the ATG site of Obp83g-2 was amplified as the promoter region. It was ligated to the pGL3-Basic (Promega) vector by cloning. The online website ALLGEN (https://alggen.lsi.upc.es/recerca/frame-recerca.html) and JASPAR with the “Insecta” group were used to predict the putative TF sites. Then, various promoter truncations of Obp83g-2 were obtained using the full promoter plasmid as the template. The over-expression constructs of TFs were produced by cloning the ORF into the pIB/V5 vector (Promega). The pIB/V5 vector was as negative control. All primers are displayed in Supplementary Table 1. The Sf9 cells were cultured in 24-well cell plates at 2 × 106 cells density. When we tested 5′-flanking regions activity, 1 μg promoter constructs and 0.02 μg PR-TK were co-transfected to the cells with the help of 2 μL of FuGENE® HD Transfection Reagent (Promega). When we tested TFs activity, 1 μg promoter constructs (0.5 μg promoter constructs and 0.5 μg TF constructs) and 0.02 μg pRL-TK (Promega) were co-transfected into the cells with the help of 2 μL of FuGENE® HD Transfection Reagent (Promega). Finally, the cells were collected after 48 h. Luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega) and TriStar2 LB 942 (Berthold, Bad Wildbad, Germany) followed protocol. Construct luciferase activity was normalized to Renilla luciferase activity.

Electrophoretic mobility shift assay

A 5′-biotinylated oligonucleotide (Supplementary Table 1) was used as the probe. Recombinant ADF-1-like protein was expressed with a His-tag, and the fusion protein was detected in the supernatant of E. coli BL21 (DE3) competent cells. The probe was incubated with the recombinant ADF-1-like protein at room temperature for 30 min. For the competing control system, recombinant His-ADF-1-like protein was incubated with 10-fold, 20-fold, and 50-fold unlabeled probes in the reaction mixture. Moreover, the His-ADF-1-like protein was also incubated with the mutant probe as a control. Reaction mixtures were loaded onto polyacrylamide gels. After electrophoresis, protein-DNA complexes were electroblotted onto transferred onto presoaked nylon membranes (Thermo Fisher Scientific) and crosslinked. Bands were visualized, and images were captured by ChemiDoc XRS (Bio-Rad Laboratories).

RNA interference

The primers (Supplementary Table 1) for dsRNA synthesis were designed using Primer 3.0. Then, the dsRNA segments were amplified and cloned by the specific dsRNA primers. The dsRNA was synthesized in vitro based on the manufacturer’s instructions of TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific). The quality and concentration of dsRNA were determined by agarose gel electrophoresis and Nanodrop One (Thermo Fisher Scientific). To enhance the efficiency of silencing, 2 μg dsRNA was injected at the end of the adult abdomen with a Nanoject II Auto-Nanoliter Injector (Drummond Scientific, Broomall, PA, USA). The 8 d females were selected for the first injection, followed by the second injection with a 24 h interval. The silencing efficiency was calculated 24 h after the second injection by comparing the expression level of selected Adf-1-like in dsAdf-1-like and double-stranded green fluorescent protein (dsGFP) injected flies. The expression of Obp83g-2 was also calculated 24 h after the second injection. Behavior assay was tested between treatment group and control group followed above described.

Statistical analysis

The statistical significance of differences between samples was analyzed using an independent t-test, and ANOVA with Tukey’s HSD post hoc test with SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The binding affinity (Kd) was defined using NanoTemper analysis software v2.3.0. All quantitative data are reported as means ± standard errors of the mean (SEM) from at least three independent experiments.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information (2.8MB, pdf)
42003_2025_8724_MOESM2_ESM.pdf (74.5KB, pdf)

Description of Additional Supplementary Files

Supplementary Data (26.7KB, xlsx)
Supplementary Movie 1 (15.7MB, mp4)
Supplementary Movie 2 (17.1MB, mp4)
Reporting Summary (2.4MB, pdf)

Acknowledgements

This work was supported by funding from National Key R&D Programmes of China (2022YFC2601000), National Natural Science Foundation of China (U21A20222, 32072491, 32302342), and China Agriculture Research System (CARS- 26).

Author contributions

H.J. and J.W. designed the research; X.C. performed prokaryotic expression, gene editing and phenotypic analysis; Q.L. reared mutant flies and performed binding analysis in vitro; C.L. screened mutant flies and performed RT-qPCR; X.C. analyzed the data; X.C. and H.J. drafted the manuscript; J.W. modified the manuscript; H.J. and J.W. provided the reagents and supervised the research; all authors read and approved the final manuscript.

Peer review

Peer review information

Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Xiaoling Xu, Michele Repetto and David Favero.

Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Data). Uncropped blot and gel images are included in Supplementary Figs. 7 and 8.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

JinJun Wang, Email: wangjinjun@swu.edu.cn.

Hongbo Jiang, Email: jhb8342@swu.edu.cn.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-025-08724-2.

References

  • 1.Leal, W. S. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu. Rev. Entomol.58, 373–391 (2013). [DOI] [PubMed] [Google Scholar]
  • 2.Duan, S. et al. NlugOBP8 in Nilaparvata lugens involved in the perception of two terpenoid compounds from rice plant. J. Agric. Food Chem.70, 16323–16334 (2022). [DOI] [PubMed] [Google Scholar]
  • 3.Hu, K., Liu, S., Qiu, L. & Li, Y. Three odorant-binding proteins are involved in the behavioral response of Sogatella furcifera to rice plant volatiles. PeerJ7, e6576 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Li, E. et al. Involvement of Holotrichia parallela odorant-binding protein 3 in the localization of oviposition sites. Int. J. Biol. Macromol.242, 124744 (2023). [DOI] [PubMed] [Google Scholar]
  • 5.Zhao, S. et al. The odorant-binding protein genes obp67 and obp56d-like encode products that guide oviposition site selection in the Asian tiger mosquito, Aedes albopictus.Insect Sci.32, 991–1003 (2024). [DOI] [PMC free article] [PubMed]
  • 6.Pelletier, J., Guidolin, A., Syed, Z., Cornel, A. J. & Leal, W. S. Knockdown of a mosquito odorant-binding protein involved in the sensitive detection of oviposition attractants. J. Chem. Ecol.36, 245–248 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Li, F. et al. Discrimination of oviposition deterrent volatile β-Ionone by odorant-binding proteins 1 and 4 in the Whitefly Bemisia tabaci. Biomolecules9, 563 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Paula, D. P. et al. Systemic and sex-biased regulation of OBP expression under semiochemical stimuli. Sci. Rep.8, 6035 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Koerte, S. et al. Evaluation of the DREAM technique for a high-throughput deorphanization of chemosensory receptors in Drosophila. Front. Mol. Neurosci.11, 366 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Khan, M. A. M. et al. Raspberry ketone diet supplement reduces attraction of sterile male Queensland fruit fly to cuelure by altering expression of chemoreceptor genes. Sci. Rep.11, 17632 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Yin, J. et al. Functional characterization of odorant-binding proteins from the scarab beetle Holotrichia oblita based on semiochemical-induced expression alteration and gene silencing. Insect Biochem. Mol. Biol.104, 11–19 (2019). [DOI] [PubMed] [Google Scholar]
  • 12.Diallo, S. et al. Antennal enriched odorant binding proteins are required for odor communication in Glossina f. fuscipes. Biomolecules11, 541 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Chen, X. et al. CRISPR/Cas9 mutagenesis abolishes odorant-binding protein BdorOBP56f-2 and impairs the perception of methyl eugenol in Bactrocera dorsalis (Hendel). Insect Biochem. Mol. Biol.139, 103656 (2021). [DOI] [PubMed] [Google Scholar]
  • 14.Gupta, B. P. & Rodrigues, V. Atonal is a proneural gene for a subset of olfactory sense organs in Drosophila. Genes Cells2, 225–233 (2003). [DOI] [PubMed] [Google Scholar]
  • 15.Komiyama, T., Johnson, W. A., Luo, L. & Jefferis, G. S. X. E. From lineage to wiring specificity: POU domain transcription factors control precise connections of Drosophila olfactory projection neurons. Cell112, 157–167 (2003). [DOI] [PubMed] [Google Scholar]
  • 16.Nair, I. S., Rodrigues, V., Reichert, H. & VijayRaghavan, K. The zinc finger transcription factor Jing is required for dendrite/axonal targeting in Drosophila antennal lobe development. Dev. Biol.381, 17–27 (2013). [DOI] [PubMed] [Google Scholar]
  • 17.Yan, H. et al. Evolution, developmental expression and function of odorant receptors in insects. J. Exp. Biol.223, jeb208215 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Chen, Y. et al. FoxO directly regulates the expression of odorant receptor genes to govern olfactory plasticity upon starvation in Bactrocera dorsalis. Insect Biochem. Mol. Biol.153, 103907 (2023). [DOI] [PubMed] [Google Scholar]
  • 19.Li, D. et al. BarH1 regulates odorant-binding proteins expression and olfactory perception of Monochamus alternatus Hope. Insect Biochem. Mol. Biol.140, 103677 (2022). [DOI] [PubMed] [Google Scholar]
  • 20.Duan, S. et al. Homeotic protein distal-less regulates NlObp8 and NlCsp10 to impact the recognition of linalool in the brown planthopper Nilaparvata lugens. J. Agric. Food Chem.71, 10291–10303 (2023). [DOI] [PubMed] [Google Scholar]
  • 21.Chen, P., Wu, W. & Hsu, J. Detection of male oriental fruit fly (Diptera: Tephritidae) susceptibility to naled- and fipronil-intoxicated Methyl Eugenol. J. Econ. Entomol.112, 316–323 (2019). [DOI] [PubMed] [Google Scholar]
  • 22.Choi, K. S., Samayoa, A. C., Hwang, S., Huang, Y. & Ahn, J. J. Thermal effect on the fecundity and longevity of Bactrocera dorsalis adults and their improved oviposition model. PLoS ONE15, e0235910 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Li, H. et al. Egg-surface bacteria are indirectly associated with oviposition aversion in Bactrocera dorsalis. Curr. Biol.30, 4432–4440 (2020). [DOI] [PubMed] [Google Scholar]
  • 24.He, M. et al. Gut bacteria induce oviposition preference through ovipositor recognition in fruit fly. Commun. Biol.5, 973 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Jayanthi, P. D. K., Kempraj, V., Aurade, R. M. & Bruce, T. J. A. Evaluation of synthetic oviposition stimulants to enhance egg collection of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae). J. Pest Sci.90, 781–786 (2017). [Google Scholar]
  • 26.Jayanthi, P. D. K., Woodcock, C. M., Caulfield, J., Birkett, M. A. & Bruce, T. J. A. Isolation and identification of host cues from mango, Mangifera indica, that attract gravid female oriental fruit fly, Bactrocera dorsalis. J. Chem. Ecol.38, 361–369 (2012). [DOI] [PubMed] [Google Scholar]
  • 27.Kempraj, V., Park, S. J. & Taylor, P. W. γ-Octalactone, an effective oviposition stimulant of Bactrocera tryoni. J. Appl. Entomol.143, 1205–1209 (2019). [Google Scholar]
  • 28.Yong, T. et al. Transcriptome analysis of female oriental fruit fly Bactrocera dorsalis under the stimulation of two oviposition attractants. J. Plant Prot.48, 1438–1446 (2021). [Google Scholar]
  • 29.Wu, H. et al. Transcriptome analysis of antennal cytochrome P450s and their transcriptional responses to plant and locust volatiles in Locusta migratoria. Int. J. Biol. Macromol.149, 741–753 (2020). [DOI] [PubMed] [Google Scholar]
  • 30.Mappin, F., Bellantuono, A. J., Ebrahimi, B. & DeGennaro, M. Odor-evoked transcriptomics of Aedes aegypti mosquitoes. Plos One18, e0293018 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Zhang, Y., Shen, C., Xia, D., Wang, J. & Tang, Q. Characterization of the Expression and Functions of Two Odorant-Binding Proteins of Sitophilus zeamais Motschulsky (Coleoptera: Curculionoidea). Insects10, 409 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Yao, R. X. et al. Characterization of the binding ability of the odorant binding protein BminOBP9 of Bactrocera minax to citrus volatiles. Pest Manag. Sicence77, 1214–1225 (2020). [DOI] [PubMed]
  • 33.Zhu, G. et al. Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing. Insect Biochem. Mol. Biol.75, 1–9 (2016). [DOI] [PubMed] [Google Scholar]
  • 34.Zhu, G. et al. CRISPR/Cas9 mediated gene knockout reveals a more important role of PBP1 than PBP2 in the perception of female sex pheromone components in Spodoptera litura. Insect Biochem. Mol. Biol.115, 103244 (2019). [DOI] [PubMed] [Google Scholar]
  • 35.Dong, X. et al. CRISPR/Cas9-mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis. Insect Sci.26, 388–399 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Jing, D. et al. GOBP1 plays a key role in sex pheromones and plant volatiles recognition in yellow peach moth, Conogethes punctiferalis (Lepidoptera: Crambidae). Insects10, 302 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Chen, X. et al. BdorOBP69a is involved in the perception of the phenylpropanoid compound methyl eugenol in oriental fruit fly (Bactrocera dorsalis) males. Insect Biochem. Mol. Biol.147, 103801 (2022). [DOI] [PubMed] [Google Scholar]
  • 38.Rachinger, N. et al. Loss of Gene Information: Discrepancies between RNA Sequencing, cDNA Microarray, and qRT-PCR. Int. J. Biol. Macromol.22, 9349 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.England, B. P., Admon, A. & Tjian, R. Cloning of Drosophila transcription factor Adf-1 reveals homology to Myb oncoproteins. Proc. Natl. Acad. Sci. USA89, 683–687 (1992). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Timmerman, C. et al. The Drosophila transcription factor Adf-1 (nalyot) regulates dendrite growth by controlling FasII and Staufen expression downstream of CaMKII and neural activity. J. Neurosci.33, 11916–11931 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.DeZazzo, J. et al. nalyot, a mutation of the Drosophila Myb-related Adf1 transcription factor, disrupts synapse formation and olfactory memory. Neuron27, 145–158 (2000). [DOI] [PubMed] [Google Scholar]
  • 42.Xu, L. et al. CRISPR-mediated mutagenesis of the odorant receptor co-receptor (Orco) gene disrupts olfaction-mediated behaviors in Bactrocera dorsalis. Insect Sci.29, 1275–1286 (2022). [DOI] [PubMed] [Google Scholar]
  • 43.Kawai, F. & Miyachi, E. -i. Ion channels and action potentials in olfactory receptor cells. Jpn. J. Psychopharmacol.27, 201–205 (2007). [PubMed] [Google Scholar]
  • 44.Dibattista, M. & Reisert, J. The odorant receptor-dependent role of olfactory marker protein in olfactory receptor neurons. J. Neurosci.36, 2995–3006 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Li, R., Ben-Chaim, Y., Yau, K. & Lin, C. Cyclic-nucleotide–gated cation current and Ca2+-activated Cl current elicited by odorant in vertebrate olfactory receptor neurons. Proc. Natl. Acad. Sci. USA113, 11078–11087 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods25, 402–408 (2001). [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information (2.8MB, pdf)
42003_2025_8724_MOESM2_ESM.pdf (74.5KB, pdf)

Description of Additional Supplementary Files

Supplementary Data (26.7KB, xlsx)
Supplementary Movie 1 (15.7MB, mp4)
Supplementary Movie 2 (17.1MB, mp4)
Reporting Summary (2.4MB, pdf)

Data Availability Statement

All data generated or analyzed during this study are included in this published article (and its Supplementary Data). Uncropped blot and gel images are included in Supplementary Figs. 7 and 8.

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