Materials
Chemicals
1-octen-3-ol (Mw = 128.21, purity 98.0%) was obtained from Yuanye Technology Co., Ltd. (Shanghai, China); trans-2-hexenal (Mw = 98.14, purity 98.0%) from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); linalool (Mw = 154.25, purity 96.0%) from Tokyo Chemical Industry Development Co., Ltd. (Shanghai, China); and anethole (Mw = 148.2, purity 98.0%) from MacLean Biochemical Technology Co., Ltd. (Shanghai, China). PHB (EM 5500 F; Mw = 750,000) was obtained from Shenzhen Ecomann Biotechnology Co., Ltd. (Shenzhen, China). PCL (Mw = 80,000) was obtained from Wokai Biotechnology Co., Ltd. (Beijing, China). PEO (Mw = 1,000,000) was obtained from Wokai Biotechnology Co., Ltd. (Beijing, China). Analytically pure chloroform was obtained from Beijing Reagent Factory. Dichloromethane (DCM) was purchased from Beijing Ouhe Technology Co., Ltd. (Beijing, China). The traps and commercially available polyethylene slow-release bottles (RS) used in the field experiments were obtained from Pherobio Technology Co., Ltd. (Beijing, China).
Insects
The L. sticticalis population used in this experiment was a multi-generational population that was laboratory-reared. Larvae were placed in a glass flask and provided with fresh C. album L. regularly. The adult L. sticticalis were kept in a cage containing 10% honey water as a source of nutrition. The rearing conditions included a temperature of 25 ± 2 °C, a relative humidity of 75 ± 10%, and a photoperiod of L16:D8h.
Preparation of food attractant-loaded fiber mat
The core-shell fiber mats (FF), capable of loading food attractants, were prepared through electrospinning using an ET-2535X system (UCALERY Development Co., Ltd., Beijing, China). The electrospinning apparatus consisted of a power supply, a syringe pump, and a collector. Based on exploration of the spinning solution formulations (Figure S1), it was determined that 8% (w/v) PHB and 2% (w/v) PCL dissolved in chloroform served as the shell spinning solution. The core spinning solution consisted of 0.5% (w/v) PEO dissolved in DCM, with the addition of 100 mg of food attractant per mL of core solution. The food attractant comprised 1-octen-3-ol, trans-2-hexenal, linalool, and anethole in a ratio of 10:5:1:1. Both solutions were stirred at room temperature at a speed of 800 rpm for at least 8 h. Additionally, fluorescein isothiocyanate dye and rhodamine B were added to the shell and core solution at a concentration of 0.01 mg/mL, respectively, for the preparation of fluorescently labeled FF. The PHB/PCL mat was prepared by uniaxial electrospinning of the shell solution for subsequent comparative experiments.
The experimental parameters for coaxial electrospinning were as follows: the outer and inner diameters of the coaxial needles were Φext/Φint = 1.8/1.3 mm for the external needle, and Φext/Φint = 0.9/0.6 mm for the internal needle. The flow rates of the shell and core solutions were 0.38 mL/h and 0.08 mL/h, respectively. The applied positive voltage was 13.5 kV, with a negative voltage of 4 kV, and the distance between the needle and the collector was set to 25 cm. The temperature was maintained at 25 ± 3 °C, and the humidity was controlled at 45 ± 2%. The prepared mats were dried at room temperature (25 ± 2 °C) for 24 h before characterization.
Characterization of food attractant-loaded fiber mat
Fourier transform infrared spectroscopy (FTIR, NICOLET 6700, Thermo Scientific, Waltham, MA, USA) was conducted with a resolution of 4 cm− 1, within a wavenumber range of 600–4000 cm− 1, and 64 scans. X-ray diffraction (XRD, Bruker D8 Advance, Bruker, Karlsruhe, Germany) was performed using Cu Kα radiation, with a 2θ range of 10–60°. X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA) employed an Al Kα X-ray source (λ = 8.33 Å), using the C 1s peak at 284.8 eV as the reference signal for semi-quantitative analysis of the surface’s external layers. An ultraviolet spectrophotometer (Shimadzu, UV-1800, Tokyo, Japan) was used to measure the absorbance of the fiber mat. Research-grade microscope (Olympus IX83, Olympus, Tokyo, Japan) was used to observe the morphology of the fiber mat under different light source voltages. Scanning electron microscopy (SEM, SU8010, Hitachi Ltd., Tokyo, Japan) was conducted at a working voltage of 10 kV, with a gold coating thickness of 8 nm. Fiber diameters were measured using ImageJ software, with the average diameter calculated across samples (n ≥ 100). Transmission electron microscopy (TEM, JEM-2100Plus, JEOL, Japan) was carried out at an electron beam accelerating voltage of 80 kV. The fiber samples for TEM observation were collected in the spinning path using a copper mesh with a carbon film, and the collection time was about 3–5 s. Laser confocal microscopy (LSM 980, Zeiss, Germany) was used to examine the fluorescently labeled FF. Thermogravimetric analysis (TGA, PerkinElmer STA 8000, PerkinElmer, Waltham, USA) was performed to evaluate the thermal stability and decomposition behavior of FF and its components. The analysis was conducted by heating from 40 °C to 800 °C at a rate of 10 °C/min under a nitrogen flow of 20 mL/min. The derivative thermogravimetry (DTG) curve was obtained by differentiating the TGA curve. The thickness of the tested fiber mats was 0.05 mm, and all tests were repeated a minimum of three times.
Loading and release performance
The loading and release performance of fiber mats are key factors determining their potential for application as food attractant carriers. First, 50 mg of the FF was weighed into a 5 mL volumetric flask, followed by the addition of methanol. The mixture was then ultrasonicated for 30 min, after which the encapsulation and loading efficiencies were analyzed using high-performance liquid chromatography (HPLC). To further evaluate the stability of the attractant in FF, the retention rates of samples stored at 4 °C, 25 °C, and 45 °C for 10 days were tested in a light-proof and sealed environment.
To evaluate the release performance of the FF, it was cut into 30 mg pieces and placed in a polyethylene centrifuge tube with an approximate diameter of 1.5 cm. The release experiment was conducted in a fume hood at room temperature under constant medium-speed ventilation conditions, with FF released naturally. Sampling was conducted at various time points, and the residual attractant content was quantified using HPLC and the external standard method. The method for extracting food attractants from fiber mats involved placing 30 mg of FF in a volumetric flask with methanol. After ultrasonic treatment for 30 min, the volume was adjusted to 5 mL, and 1 mL of the solution was filtered into an injection bottle for analysis. To analyze the release mechanism of food attractants in FF, various release kinetic models were employed to fit and evaluate the cumulative release rates at different time points. These models included the zero-order model, first-order model, Higuchi model, Ritger-Peppas model, and Weibull model, all of which aimed to reveal the release mechanism.
The HPLC test conditions were as follows: HPLC (1200-DAD, Agilent, Santa Clara, CA, USA) analysis utilized a ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm). The mobile phase for 1-octen-3-ol was methanol: water (v/v) = 60:40, with a flow rate of 1.0 mL/min, an injection volume of 10 µL, a column temperature of 30 °C, and a diode array detector signal at 226 nm. The mobile phase for trans-2-hexenal was methanol: water (v/v) = 60:40, with a flow rate of 1.0 mL/min, an injection volume of 10 µL, a column temperature of 30 °C, and a diode array detector signal at 260 nm. The mobile phase for linalool was acetonitrile: water (v/v) = 55:45, with a flow rate of 1.0 mL/min, an injection volume of 10 µL, a column temperature of 25 °C, and a diode array detector signal at 210 nm. The mobile phase for anethole was acetonitrile: water (v/v) = 20:80, with a flow rate of 1.0 mL/min, an injection volume of 10 µL, a column temperature of 25 °C, and a diode array detector signal at 254 nm.
Stress resistance and degradability
The water contact angle meter (OCA 20, Data Physics, Germany) was used to measure the water contact angle (WCA) of the fiber mat. A 2 µL water droplet was placed on the FF surface, and the change in WCA was recorded over a period of 30 min until the droplet spread completely. The effect of humidity on FF was assessed by controlling humidity levels (100% and 50%) in an artificial climate chamber. Swelling rate: FF was immersed in water, and the sample was removed after 1 h, 6 h, 12 h, and 24 h. Excess surface water was gently absorbed with filter paper, and the weight was immediately measured. Solubility rate: FF was placed in a beaker and stirred at 500 rpm and 25 °C for solubility. The residual fiber mat was removed after 12 h, 24 h, 36 h, and 48 h, and weighed after ensuring it was completely dry. The swelling/solubility rate was calculated as follows:
$$\begin{aligned}&Swelling/solubility\:rate\:\left(\%\right)\cr&\quad=\frac{Weight\:after\:test-Initial\:weight}{Initial\:weight}\end{aligned}$$
(1)
The reflectance of the FF was measured using a UV-vis-Infrared spectrophotometer (Hitachi, UH4150, Tokyo, Japan). The mats were cut to dimensions of 10 × 30 × 0.05 mm and reflectance was recorded over a wavelength range of 200–800 nm. All tests were repeated 3–5 times, and mean values were calculated for statistical analysis. UV weathering test: 50 mg of FF and filter paper (as a control) were weighed and placed in a UV aging box for 1 day, after which their retention rates were calculated of attractant. The light source was a xenon arc lamp with a wavelength of 340 nm and an irradiance of 0.76 W/m2/nm. The exposure cycle consisted of 8 h of drying followed by 4 h of condensation.
To evaluate the mechanical properties of FF, they were measured by standard tensile test method. FF was cut into 25 mm × 13 mm pieces and subjected to tensile testing on a universal testing machine (Instron, Canton, MA). The grip spacing was set to 50 mm. During the test, the tensile stress-strain curves of the samples were recorded and compared with the PHB/PCL mats to analyze the differences in mechanical properties caused by the material composition.
Composting experiments were conducted on FF and RS for comparison. The samples were placed in soil with controlled humidity of 15%, temperature of 25℃ and the soil was silty loam (44.39% sand, 51.05% silt, and 4.56% clay) with an organic matter content of 16.75%. The samples were taken out and weighed at 1, 3, 5, and 7 days, and the degradation rate was calculated according to the formula reported previously [39]. The changes in the morphology and chemical properties of FF at different time points were examined using SEM and FTIR, respectively. To evaluate the degradation performance of the FF, mats were regularly collected and observed throughout the field trial. In addition, FF was placed in a field environment to simulate actual application scenarios, and changes in its diameter were examined using SEM to further analyze its degradation behavior comprehensively.
Biological activity assay
Electroantennogram (EAG) recording
Insect antennae are crucial structures for receiving and processing external chemical signals. To investigate the electrophysiological response of L. sticticalis antennae to FF, we used an EAG IDAC 4 system (Syntech, Buchenbach, Germany) to test the antennal potential responses of both male and female adults to freshly prepared FF. Specifically, the antennae were rapidly excised using a scalpel, and the antennal base was connected to the negative electrode using SPECTRA 360 electrode gel (Parker Lab, USA), while the tip was connected to the positive electrode. For each EAG recording, 200 mg of freshly prepared FF was folded into a rectangular shape (2 cm in length and 0.5 cm in width) and placed in a Pasteur pipette as the stimulus source after the solvent had evaporated. FF without attractant were used as the control. The stimulus, placed in the Pasteur pipette, was injected into a charcoal-filtered and humidified airflow for 0.2 s at a flow rate of 500 mL/min, delivered to the antennae by an air stimulus controller CS-55 (Syntech, Germany).
The antennal responses to the volatiles from the FF were recorded using a PRG-3 probe. The signals were processed with an IDAC-4 data acquisition controller and analyzed using EAG Pro software (Syntech, Germany). Additionally, we periodically tested the changes in electrophysiological responses of both male and female L. sticticalis to FF with varying release times under a fume hood. Each experiment was conducted with three biological replicates and three technical replicates.
Behavioral tests
The olfactory behavioral responses of both male and female adults of the L. sticticalis were evaluated using an insect olfactory behavior selection apparatus (ZL2023204533809) to assess their indoor behavioral responses toward the FF. The olfactory behavior detection apparatus consists of a gas purification and humidification system, an atmospheric sampler, a core storage chamber, an odor chamber, a behavior detection unit, and an observation device. Prior to the bioassays, the main behavioral detection unit was cleaned with ethanol and water, and the atmospheric sampler was operated for 15 min to remove any residual odors. Subsequently, 3-day-old L. sticticalis adults were acclimated in the testing room for 15 min, while 200 mg of freshly prepared FF, from which the solvent had evaporated, were placed in the odor chamber as the odor source. Another chamber containing FF without attractant served as the control. The behavior of the insects was observed by recording the number of insects found in the odor and control chambers.
The effect of FF with different release times on insect attraction was periodically tested under a fume hood. Each experiment included three replicates, with each replicate consisting of 30 male or female L. sticticalis adults. The insect behavior selection rate was calculated using Formula (2):
$$\begin{aligned}&{\rm{selection}}\,rate\,(\% ) \cr&\quad= {{Number\,of\,in{\rm{sec}}ts\,choosing\,the\,odor\,chamber} \over \matrix{ Number\,of\,in{\rm{sec}}ts\,choosing\,the\,odor\,chamber\, \hfill \cr + Number\,of\,in{\rm{sec}}ts\,choosing\,the\,control\,chamber \hfill \cr} }\end{aligned}$$
(2)
Trapping performance
During the L. sticticalis infestation period, field trapping experiments using FF loaded with food attractants were conducted in a Pisum sativum field located in Kangbao County, Zhangjiakou City, Hebei Province, China (41°52’3.77″N, 114°36’16.28″E). In this experiment, the attractant loading in the FF was 1 mg, and RS loaded with the same amount of attractant was used as the control group, with empty traps serving as the blank control (CK). Each sample was tested with six replicates. Freshly prepared FF were cut into strips and wrapped around both sides of the trap slots, while the commercial carriers were installed in the ring slots of the traps. The distance between traps was approximately 20 m. The positions of the traps were changed regularly to eliminate the interference of location factors on trapping. Trapping data were collected regularly, and pests were removed from the traps. Neither the attractants nor the traps were replaced during the entire experimental period.
Statistical analysis
OriginLab software was used to process data and curve fitting. For comparisons across multiple groups, one-way analysis of variance (ANOVA) was followed by Duncan’s multiple range test. Different lowercase letters indicate significant differences between datasets, while *P < 0.05 denotes levels of statistical significance. ns indicates no significant difference. Each dataset is based on at least three independent experiments.
