Molecular Characterization and Expression Patterns of Two Pheromone-Binding Proteins from the Diurnal Moth Phauda flammans (Walker) (Lepidoptera: Zygaenoidea: Phaudidae)

Sex pheromone-binding proteins (PBPs) play an important role in sex pheromone recognition in Lepidoptera. However, the mechanisms of chemical communication mediating the response to sex pheromones remain unclear in the diurnal moths of the superfamily Zygaenoidea. In this study, Phauda flammans (Walker) (Lepidoptera: Zygaenoidea: Phaudidae) was used as a model insect to explore the molecular mechanism of sex pheromone perception in the superfamily Zygaenoidea. Two novel pheromone-binding proteins (PflaPBP1 and PflaPBP2) from P. flammans were identified. The two pheromone-binding proteins were predominantly expressed in the antennae of P. flammans male and female moths, in which PflaPBP1 had stronger binding affinity to the female sex pheromones Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal, PflaPBP2 had stronger binding affinity only for (Z, Z, Z)-9, 12, 15-octadecatrienal, and no apparent binding affinity to Z-9-hexadecenal. The molecular docking results indicated that Ile 170 and Leu 169 are predicted to be important in the binding of the sex pheromone to PflaPBP1 and PflaPBP2. We concluded that PflaPBP1 and PflaPBP2 may be responsible for the recognition of two sex pheromone components and may function differently in female and male P. flammans. These results provide a foundation for the development of pest control by exploring sex pheromone blocking agents and the application of sex pheromones and their analogs for insect pests in the superfamily Zygaenoidea.


Introduction
Moths detect pheromones with specialized chemosensilla localized on the antennae or, more rarely, on other parts of the body [1]. Normally, moth antennae can accurately identify and distinguish sex pheromones and their analogs by pheromone-binding proteins (PBPs) [2]. PBPs are either specific to or highly enriched in the antennae of males and act as pheromone carriers by binding and transporting odorant molecules across the antennal hemolymph to odorant receptor proteins [3,4]. Some studies have shown that the sexually dimorphic antennal flagellum of the male is enlarged so that the flagella can house extra arrays of pheromone-specific long trichoid sensilla, where PBPs have high binding affinities to sex pheromones released by female moths [5,6]. Further studies indicated that the female moth antennae can also detect their conspecific sex pheromones [7], and the expression of PBPs has been observed in antennae of female moths such as Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae), Antheraea polyphemus (Cramer) (Lepidoptera: Saturniidae), and Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae) [8][9][10]. Previous studies have

Tissue Expression of PflaPBPs
The expression levels of PflaPBP1 and PflaPBP2 varied significantly among adult tissues ( Figure 2, Tables 1 and 2). Specifically, expression levels of PflaPBP1 and PflaPBP2 in the antennae were 241.17 times and 267.38 times the sum of other tissues, respectively. Differences in PflaPBP1 expression were also found between sexes (Table  1). There were significant interactions between sex and tissues, showing that PflaPBP1 and PflaPBP2 are expressed at different levels in the same tissue in different sexes (Tables 1 and 2). In particular, the expression of PflaPBP1 was 1.56 times higher in the antennae of males than in those of females, and the expression of PflaPBP2 was 1.57 times higher in the antennae of females than in those of males ( Figure 2).

Tissue Expression of PflaPBPs
The expression levels of PflaPBP1 and PflaPBP2 varied significantly among adult tissues ( Figure 2, Tables 1 and 2). Specifically, expression levels of PflaPBP1 and PflaPBP2 in the antennae were 241.17 times and 267.38 times the sum of other tissues, respectively. Differences in PflaPBP1 expression were also found between sexes (Table 1). There were significant interactions between sex and tissues, showing that PflaPBP1 and PflaPBP2 are expressed at different levels in the same tissue in different sexes (Tables 1 and 2). In particular, the expression of PflaPBP1 was 1.56 times higher in the antennae of males than in those of females, and the expression of PflaPBP2 was 1.57 times higher in the antennae of females than in those of males ( Figure 2).

Prokaryotic Expression and Purification of PflaPBPs
Recombinant proteins were induced and expressed in E. coli prokaryotic cells in vitro. After induction by isopropyl-β-D-1-thiogalactopyranoside (IPTG), there were obvious protein bands between 25 and 35 kDa in the experimental group compared with the control group, indicating that the protein was successfully induced. Then, the histidine (His) tag was removed and purified to obtain the target soluble protein from the supernatant (Figure 3). The yield of purified protein was 0.21 mg/mL for PflaPBP1 and 0.07 mg/mL for PflaPBP2.

Prokaryotic Expression and Purification of PflaPBPs
Recombinant proteins were induced and expressed in E. coli prokaryotic cells in vitro. After induction by isopropyl-β-D-1-thiogalactopyranoside (IPTG), there were obvious protein bands between 25 and 35 kDa in the experimental group compared with the control group, indicating that the protein was successfully induced. Then, the histidine (His) tag was removed and purified to obtain the target soluble protein from the supernatant ( Figure 3). The yield of purified protein was 0.21 mg/mL for PflaPBP1 and 0.07 mg/mL for PflaPBP2.

Test of Fluorescent Probe
To detect the binding affinity of proteins to small molecules by fluorescence competitive binding, N-phenyl-1-naphthylamine (1-NPN) was used as the probe to test the suitability of PflaPBP1 and PflaPBP2 at pH = 7.4. The maximum fluorescence value of the complex of the two proteins and 1-NPN increased gradually with the increase in the concentration of 1-NPN until it reached saturation. Scatchard plots show that 1-NPN binds the two proteins one-to-one under neutral conditions ( Figure 4). Thus, 1-NPN can be used as a probe for fluorescence competitive binding experiments of PflaPBP1 and PflaPBP2.

Test of Fluorescent Probe
To detect the binding affinity of proteins to small molecules by fluorescence competitive binding, N-phenyl-1-naphthylamine (1-NPN) was used as the probe to test the suitability of PflaPBP1 and PflaPBP2 at pH = 7.4. The maximum fluorescence value of the complex of the two proteins and 1-NPN increased gradually with the increase in the concentration of 1-NPN until it reached saturation. Scatchard plots show that 1-NPN binds the two proteins one-to-one under neutral conditions ( Figure 4). Thus, 1-NPN can be used as a probe for fluorescence competitive binding experiments of PflaPBP1 and PflaPBP2.

Test of Fluorescent Probe
To detect the binding affinity of proteins to small molecules by fluorescence competitive binding, N-phenyl-1-naphthylamine (1-NPN) was used as the probe to test the suitability of PflaPBP1 and PflaPBP2 at pH = 7.4. The maximum fluorescence value of the complex of the two proteins and 1-NPN increased gradually with the increase in the concentration of 1-NPN until it reached saturation. Scatchard plots show that 1-NPN binds the two proteins one-to-one under neutral conditions ( Figure 4). Thus, 1-NPN can be used as a probe for fluorescence competitive binding experiments of PflaPBP1 and PflaPBP2.

Fluorescence Binding Affinities
The binding characteristics of PflaPBP1 and PflaPBP2 with two sex pheromones were analyzed by fluorescence competitive binding experiments ( Figure 5). These results showed that the binding affinity of PflaPBP1 to ligands was bound with a higher affinity than the binding affinity of PflaPBP2 to ligands. PflaPBP1 had a moderate binding affinity for the female sex pheromones Z-9-hexadecenal (Ki = 21.26 µM) and (Z, Z, Z)-9, 12, 15octadecatrienal (Ki = 38.48 µM), and PflaPBP2 had a moderate binding affinity for (Z, Z, Z)-9, 12, 15-octadecatrienal (Ki = 33.95 µM) but no detectable binding affinity for Z-9hexadecenal (Ki = 108.68 µM) ( Table 3).

Three-Dimensional Structure and Molecular Docking of PflaPBPs
The protein structure of BmorPBP1 was used as a template to construct the protein structures of PflaPBP1 and PflaPBP2. The consistency between PflaPBP1 and PflaPBP2 with the best template was different, with 67.38% (the evaluation of Ramachandran plots was 92.67%, Figure A3) and 47.89% (the evaluation of Ramachandran plots was 95.86%, Figure A4) sequence consistency, respectively. The three-dimensional structure consists of six α-helixes and one additional α-helix (α7-helix) ( Figure 6A,B). In addition, three disulfide bonds are formed by the six conserved cysteine (Cys) residues to connect, which are α1-α3 (Cys45-Cys84), α3-α7 (Cys78-Cys142), and α6-α7 (Cys97-Cys117) in PflaPBP1 and α1-α3 (Cys50-Cys87), α3-α7 (Cys83-Cys143), and α6-α7 (Cys87-Cys152) in PflaPBP2 ( Figure 6C). The Cys sites of PflaPBP1 and PflaPBP2 are very similar to the disulfide bond. To explore the binding site of PflaPBP1 and PflaPBP2 with sex pheromones, we selected sex pheromones [Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal] that possibly bind to PflaPBP1 and PflaPBP2 for three-dimensional structural docking. The results of molecular docking indicated that hydrogen bonding was the main interaction mode between PflaPBP1/PflaPBP2 and the tested sex pheromone molecules.

Discussion
Many species of Zygaenoidea release sex pheromones which can be used in the monitoring and forecasting of these diurnal moths in the field [31,[34][35][36][37][38][39]. P. flammans sex pheromones have been used as biological control agents in the field, and a mixture of the two synthetic pheromones of P. flammans, Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15octadecatrienal, at a ratio of 1:1 could effectively attract male moths [32]. Since field trials are normally more convincing to identify sex pheromones in insects, Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal were proven to be female-produced sex pheromones that could attract males [32]. Based on our findings, we deduced that PflaPBP1, but not PflaPBP2, was the dominant binding protein in P. flammans as a passive carrier delivering sex pheromones to neuronal membranes. First, a mixture of Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal at a ratio of 1:1 was effective in attracting male moths [32], which indicated that the recognition of both chemicals was needed. Second, PflaPBP1 was in accordance with the male-biased expression in antennae (Figure 2A). In addition, PflaPBP1 has binding affinity to both sex pheromones, while PflaPBP2 could only bind Z-9-hexadecenal ( Figure 5).
Our results also indicated that PflaPBP1 and PflaPBP2 may function differently in females and males. Quantitative real-time PCR analysis of the PflaPBP1 and PflaPBP2 genes showed that they were predominantly expressed in the antennae of both sexes in P. flammans, similar to the findings in several diurnal moths, e.g., M. sexta [8], A. polyphemus [10], and B. mori [26]. Interestingly, the transcript levels of the PflaPBP genes in the antennae of both sexes were different. For example, the expression level of PflaPBP1 in male antennae was significantly higher than the expression level of PflaPBP1 in female antennae and vice versa for PflaPBP2 (Figure 2). Further analysis of the binding characteristics of PflaPBP1 with the two sex pheromones indicated an important role in female sex pheromone perception. Therefore, the male-biased expression level of PflaPBP1 suggested that the gene may be involved in sexual communication involving pheromones. For the PflaPBP2 gene, the expression level in female antennae was significantly higher than the expression level in male antennae, and the protein could bind (Z, Z, Z)-9, 12, 15-octadecatrienal. In this context, the presence of the PflaPBP2 gene in the olfactory sensilla of female antennae suggests that P. flammans females may detect either their own sex pheromone or at least certain components of their sex pheromone. There are two hypotheses to explain these results. First, females detecting sex pheromones reproduced by themselves are believed to allow the detection of competing plumes for avoidance [17,55]. Especially for the monandrous moth P. flammans, conspecific signals are used as cues for rapid relocation to valuable sites to avoid competition in their short lifespan because delayed mating significantly reduces their reproductive outputs [56]. Thus, conspecific communication with pheromones may improve the chances of mating success [57,58]. Second, female expression of PflaPBP2 may help females recognize the Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal released by other females, which is used to avoid the identification bias of males among similar species, such as P. triadum Walker (Lepidoptera: Phaudidae), leading to crossmating of different species [7]. Indeed, female autodetection of their sex pheromones has been demonstrated for several diurnal moths, including Panaxia quadripunctaria Poda (Lepidoptera: Arctiidae) [59] and Utetheisa ornatrix (Linnaeus) (Lepidoptera: Erebidae) [60]. Our results suggest that PflaPBP1 and PflaPBP2 may play different roles in female sex pheromone perception and that these genes may undergo functional differentiation.
In this study, sex pheromone components of P. flammans, Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal, were bound with PflaPBP1 and PflaPBP2 with different degrees of sensitivity. The three-dimensional structure consists of six α-helixes and one additional α-helix (α7-helix) ( Figure 6A,B), which may be involved in the binding and release of pheromones under different conditions. For example, PflaPBP1 showed binding to the female sex pheromones Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal, while PflaPBP2 showed binding only to (Z, Z, Z)-9, 12, 15-octadecatrienal ( Figure 5). Both sex pheromone ligands showed the same chain length based on the three-dimensional structure models. One end of each molecule had hydroxyl groups, which tended to form hydrogen bonds with PBP residues. These hydrogen bonds were considered to be the main interaction mode between PflaPBPs proteins and sex pheromone ligands, which has been confirmed in several insect PBPs [61,62]. Furthermore, the docking results showed that both the binding sites of PflaPBP1 and the two sex pheromone ligands involved Ile 170. We speculated that Ile 170 might be the key site for the recognition of PflaPBP1.
The preceding studies found that the binding affinity of PBPs for pheromones could be significantly decreased or totally discontinued even with the mutation of one or two amino acids [63,64]. A study also conjectured that some hydrophobic residues and their hydrophobic interactions were essential for binding to their sex pheromone components [65]. Moreover, further functional verification of PflaPBPs by RNAi survey rather than point mutation should provide direct biological evidence that was fully considered. Unfortunately, PflaPBP1 and PflaPBP2 could not be effectively depressed by RNA interference after many attempts (unpublished data). We deduced that low stability and intracellular transport of dsRNA might lead to a poor RNAi response in P. flammans, which was consistent with other lepidopterans such as Spodoptera littoralis (Boisduval) and Chrysodeixis includens (Walker) [66][67][68][69][70][71][72]. Furthermore, the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 technique as a substitutive method for RNAi would be certain to facilitate functional research of PflaPBP1 and PflaPBP2, combined with electrophysiological and behavioral assays to verify their functions [65], which cannot be implemented at present because we were unable to collect P. flammans eggs successfully due to the lower mate success in the laboratory. For this reason, we have taken transgenerational breeding in P. flammans as a priority for the prospective study of gene function via the CRISPR/Cas9 technique, which is currently achieving limited progress.
There is a possibility that other sex pheromone components participate in sex pheromone recognition in P. flammans, which must be considered. First, we note that the identification of sex pheromone components and their proportion is a process of constant and precise adjustment in many species of lepidoptera, such as Spodoptera frugiperda (J. E. Smith) [73]. Meanwhile, there is also the possibility of a novel sex pheromone component in P. flammans that might be identified in the future with new technology development. Thus far, Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal have been proved as two vital components by a series of laboratory and field studies [32]. In this context, we focus on the known sex pheromone components and their interactions with PflaPBP1 and PflaPBP2.
Only two PflaPBPs were involved in this study, while 3~4 PBPs have been identified in some lepidopteran species [74,75]. These two PflaPBPs were originally identified from an antenna Illumina high-throughput transcriptome (unpublished data) in P. flammans, and their binding affinity for Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal was also confirmed. However, we noticed that there might be more than two PflaPBPs in P. flammans just like B. mori [76]. First, a more advanced antenna transcriptome via PacBio full-length sequencing was established to find a third PflaPBP with no success [77]. Second, local basic local alignment search tool (BLAST) was also conducted, and no additional PflaPBP was found. These attempts convince us that it is very likely to have only two PflaPBPs in P. flammans. Moreover, the number of PBPs may be caused by species specificity because only two PBPs have also been reported in many insects, such as Sitotroga cerealella (Olivier) [78]. However, 58 OBPs were identified in P. flammans [77]. There is a possibility that some of them might act as a PBP in addition to PflaPBP1 and PflaPBP2 with a grain of salt because their functional studies have not yet been conducted.
Currently, studies on sex pheromone binding proteins of Lepidoptera are focused mainly on nocturnal moths, while similar studies on diurnal moths remain unclear. For the first time, we illustrated the interactions between the known sex pheromones and the identified pheromone binding proteins in vitro, which have a guiding role in probing the olfactory molecular mechanism of sex pheromone perception in the superfamily Zygaenoidea as well as other diurnal species.
In conclusion, PflaPBP1 and PflaPBP2 are responsible for the recognition of Z-9hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal to varying degrees. These findings may facilitate pest control by exploring sex pheromone blocking agents. Our research will also help with the application of sex pheromones and their analogs in the diurnal moths of superfamily Zygaenoidea.

Insect Rearing and Sample Collection
From June to July 2018, P. flammans larvae were collected on Ficus benjamina L. trees located at the campus of Guangxi University (Nanning City, Guangxi Zhuang Autonomous Region, China; 108.17 • E, 22.50 • N). The collected larvae were then routinely placed into plastic boxes (radius = 8.0 cm, height = 12 cm; 10 individuals/box) and incubated in a standardized insect rearing room. The room was set to 26 (±1) • C, with a relative humidity of 60-80% and a photoperiod of L 14: D 10. Fresh young leaves of F. benjamina were replaced daily to feed the larvae until the pupae were collected. The pupae were then sexed following Mao et al. [79] and kept individually in plastic tubes (2.7 cm diameter, 10.3 cm height) with a plastic lid with 10 holes (2 mm diameter) in the middle for ventilation. Emerged adult moths were reared with 10% sucrose solution before use.
The antennae, heads (without antennae), thoraxes, abdomens, legs, and wings were dissected from 10 living individuals by surgical scissors (Shandong Xinhua Medical Devices Co., Ltd., Zibo, China) to prepare tissue samples. The sampled tissues were frozen immediately in liquid nitrogen after collection and stored at −80 • C. Three biological repeats were prepared.

RNA Extraction and Cloning of PflaPBPs
Total RNA was extracted from samples of P. flammans using TRIzol reagent (Takara, Beijing, China). Total RNA was examined by a NanoDrop 2000 system (Thermo Scientific™, Waltham, MA, USA) and 1% agarose gel electrophoresis (Newbio, San Francisco, CA, USA). Total RNA with a clear band and an OD260/280 value of 1.9-2.1 was used for further cDNA preparation. cDNA preparation was performed in accordance with the instructions of the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Beijing, China, RR047A) and stored at −20 • C.
PBP and pheromone-binding protein were used as keywords to screen the annotated chemosensory genes by the results of Nr annotation from the antenna transcriptome database. So, candidate PBPs were identified from the antenna transcriptome database of P. flammans (unpublished data) and applied to primer design for the ORF (Table 4)  The PCR cycling conditions were as follows: initial denaturation at 95 • C for 1 min; then, 33 cycles of 95 • C for 30 s, 60 • C for 30 s, and 72 • C for 20 s; and a final extension at 72 • C for 5 min. ORF cloning was performed by PCR using polymerase. Then, the PCR product was linked to the pMD18-T vector (Takara, China) and transformed into Trans-t1 competent cells (preparation by us) according to the product manual. Sequence comparison of the cloned genes was performed. Table 4. Primers for gene cloning and qRT-PCR in P. flammans.

Sequence Analysis
The homology was first compared by the BLAST of the NCBI database, and then, the amino acid sequence of the obtained sequence was predicted and deduced by using ExPASy online protein analysis website (https://web.expasy.org/translate/, accessed on 3 November 2022). Genedoc version 2.0 software was used for protein sequence alignment. MEGA 7.0 software was subsequently used to construct the phylogenetic tree of PflaPBPs and other lepidopteran PBPs [80].

Tissue Expression of PflaPBPs
The expression levels of PflaPBPs in different tissues of adult moths were measured by qRT-PCR. GAPDH and TUB2 were used as internal reference genes [81]. Each treatment had three biological repeats and three technical repeats. The related qRT-PCR primers are listed in Table 3. Primers for qRT-PCR were evaluated before the expression evaluation with five times gradient dilution of the cDNA as DNA template to calculate PCR efficiency(E): E = 10 −1/k − 1, where k is the slope of standard curve and regression coefficient: Cq = −klgX 0 + b, where Cq is the threshold cycle, k is the slope of standard curve, X 0 is the template concentration. The PCR system (10 µL) was prepared on ice according to the following system: 5 µL of SYBR ® Premix Ex Taq™ II (Tli RNaseH Plus), 2.6 µL of ddH 2 O, 0.2 µL of each primer (forward and reverse primers, 10 µM), and 2.0 µL of cDNA template. The qRT-PCR procedure was as follows: 95 • C for 3 s; 40 cycles at 95 • C for 5 s and at 60 • C for 30 s; and melting curve analysis at 95 • C for 15 s; 60 • C for 1 min; and 95 • C for 15 s. For tissue expression of PflaPBPs, cDNA samples were all diluted 20 times, and the obtained data were normalized by the 2 −∆∆Ct method [82].

Expression and Purification
The target fragments PflaPBP1 and PflaPBP2 were subcloned into the BamHI and XhoI sites of pATX-sumo expression vectors. Single colonies containing recombinant plasmids were selected from the plate and inoculated in 5 mL of Luria-Bertani (LB) medium containing kanamycin (50 µg/mL) overnight at 37 • C. Subsequently, 200 µL was inoculated into 20 mL of LB medium containing antibiotics and incubated at 37 • C until OD 600 = 0.6. Then, the cells were transformed into T7E and BL21 strains, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added at 16 • C for 16 h. The target protein with an N-terminal 6*His tag was expressed and purified. Samples were collected, and 12,000 rpm centrifugation for 1 min was used to collect the bacteria. An ultrasound device was used to lyse bacterial cells suspended in 200 µL of phosphate-buffered saline (PBS). The ultrasound time was 2 s, and the interval was 2 s, totaling 4 min. The supernatant and precipitate were separated by centrifugation at 12,000 rpm for 2 min. The retained final PflaPBPs were in the supernatant and applied for purification by nickel column affinity chromatography (Thermo, USA) according to the manufacturer's specifications with two washes.
Recombinant bovine enterokinase was added to the eluted proteins and incubated at 26 • C for 10 h to remove the His-tags from the recombinant proteins. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (12%, 80 V concentrated gel, 20 min, 120 V separated gel, 45 min) was performed to assess the protein expression and purification. The purified protein was dialyzed in Tris buffer (pH 7.4 and pH 5.0), subpackaged and frozen in a −80 • C ultralow temperature refrigerator.

Probe Test and Fluorescence Binding Assays
An RF-5301PC fluorescence spectrophotometer (Shimadzu, Kyoto, Japan) was used in this experiment for fluorescence competitive binding analysis with a 1-cm quartz colorimetric cup at room temperature. N-Phenyl-1-naphthylamine (1-NPN) was used as the probe and applied for the suitability test. The 1-NPN was excited at 337 nm, in which the emission spectra were recorded between 350 and 660 nm. The purified PflaPBP1 and PflaPBP2 were diluted to a final concentration of 2 µM of the protein solution in Tris-HCl buffer and titrated with aliquots of 1 mM 1-NPN dissolved in methanol, which were 0, 2, 4, 8, 12, 16, 20 µM. The maximum fluorescence value (manipulate peak pick) of each excitation was recorded. The binding data were obtained from three independent measurements. The dissociation constants of PflaPBP1/PflaPBP2 for 1-NPN were calculated from Scatchard plots of the binding data by using Prism 5 software (GraphPad, La Jolla, CA, USA) with nonlinear regression for the one site-specific binding method.
The two sex pheromones Z-9-hexadecenal (Z9H, 96.3% chemical purity) and (Z, Z, Z)-9,12,15-octadecatrienal (Z9O, 93% chemical purity) were obtained from Shanghai T & W Pharmaceutical Co., Ltd. (Shanghai, China) and Pherobank BV (Wijk bij Duurstede, The Netherlands) [32]. The competitive binding of these two sex pheromone components was measured using 1-NPN (2 µM) as the fluorescent probe with a stoichiometry of 1:1 (protein: ligand). PflaPBP1 or PflaPBP2 was titrated to a final concentration of 2 µmol/L with 50 mM Tris-HCl buffer. The sex pheromone dissolved in methanol was gradually added to the mixed solution of protein and 1-NPN with the final concentration of each ligand added to the sample ranging from 0 µM to 20 µM. All sets of fluorescence spectrophotometers were the same as above. The binding data were obtained from three independent measurements. Reduction in relative fluorescence intensity indicated that the competing compound displaced 1-NPN from the binding site of PflaPBP1 or PflaPBP2. The following formula was adopted to calculate the binding affinities (Ki): Ki = [IC 50 ]  is the free concentration of 1-NPN, [IC50] is the ligand concentration replacing 50% of the fluorescence reporter, and K 1-NPN is the dissociation constant of the PflaPBP/1-NPN complex. The data were fitted with nonlinear regression model using Prism software (version 5.0).

Three-Dimensional Structures and Molecular Docking of PflaPBPs
With the SWISS MODEL website (https://swissmodel.expasy.org/interactive, accessed on 3 November 2022), PflaPBP1 and PflaPBP2 were compared to proteins in the CSBP Protein Data Bank (PDB). The selected template sequence must be more than 30% similar to the target sequence [83]. As a model insect, B. mori is the most advanced sex pheromone binding protein in the lepidopteran family. The similarity of BmorPBP (PDB ID: 1GM0.1) to PflaPBP1 and PflaPBP2 was 57.23% and 38.15%, respectively. Therefore, this BmorPBP was selected as the structural template to establish the three-dimensional structure model of PflaPBP1 and PflaPBP2.
The protein was pretreated by AutoTools and then calculated by AutoGrid. The active sites were defined as potential sites calculated by POCASA (http://altair.sci.hokudai.ac. jp/g6/service/pocasa/, accessed on 3 November 2022), and grid box coordinates were set. PflaPBP1 is (34.946, −11.031, 38.975), and the box size is 20 × 20 × 20 mesh points; PflaPBP2 is (9.028, 4.576, 32.343), the box size is 20 × 20 × 20 mesh points, and the distance of each small mesh point is 0.1 nm. AutoDock Vina was used to dock small molecules with the proteins. Finally, the dominant conformation was analyzed, and Schrodinger was used for mapping.

Statistical Analysis
Data analysis was performed using SPSS 19.0 (IBM Corp., Chicago, IL, USA). The expression of PflaPBPs was analyzed using two-way analysis of variance (ANOVA). A level of p < 0.05 was accepted as a significant difference.  Acknowledgments: We thank two anonymous reviewers for their constructive comments, which have significantly improved the article.

Conflicts of Interest:
The authors declare no conflict of interest.
Appendix A grid box coordinates were set. PflaPBP1 is (34.946, −11.031, 38.975), and the box size is 20 × 20 × 20 mesh points; PflaPBP2 is (9.028, 4.576, 32.343), the box size is 20 × 20 × 20 mesh points, and the distance of each small mesh point is 0.1 nm. AutoDock Vina was used to dock small molecules with the proteins. Finally, the dominant conformation was analyzed, and Schrodinger was used for mapping.

Statistical Analysis
Data analysis was performed using SPSS 19.0 (IBM Corp., Chicago, IL, USA). The expression of PflaPBPs was analyzed using two-way analysis of variance (ANOVA). A level of p < 0.05 was accepted as a significant difference.       Figure A3. Rationality evaluation results of the PflaPBP1 3D model. The red points indicate amino acid residues, the green and light green areas indicate permitted areas, and the white areas indicate nonpermitted areas. Figure A4. Rationality evaluation results of the PflaPBP2 3D model. The red points indicate amino acid residues, the green and light green areas indicate permitted areas, and the white areas indicate nonpermitted areas.