Plant volatile-based fumigation improves mating competitiveness of males for population suppression of the global fruit pest Cydia pomonella

Plant volatile-based fumigation improves mating competitiveness of males for population suppression of the global fruit pest Cydia pomonella | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plant volatile-based fumigation improves mating competitiveness of males for population suppression of the global fruit pest Cydia pomonella Sheng-Wang Huang, Peng-Cheng Wang, Yan Wang, Jie-Qiong Wang, Ping Gao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4677671/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The implementation of sterile insect technique (SIT) has proven effective in the area-wide suppression of several significant agricultural and sanitary pests by employing traditional cobalt-60 ( 60 Co-γ) as a radiation source. Recently, X-ray has been validated as a feasible alternative to 60 Co-γ radiation sources. Nonetheless, higher doses of X-ray irradiation lead to insect sterility but diminish mating competitiveness, thereby impacting the effectiveness of SIT applications. Thus, it is crucial to ascertain the optimal irradiation dose and develop strategies to enhance the mating competitiveness of sterile insects to enhance SIT efficacy. In this study, we determined the effect of various X-ray irradiation doses (ranging from 0 to 366 Gy) on the fecundity, fertility, and mating competitiveness of Cydia pomonella , a globally invasive fruit pest. Results demonstrated that the sterility rate of sterile males increased proportionally with irradiation dose up to 200 Gy, beyond which it plateaued. Notably, exposure to 200 Gy of irradiation notably decreased the mating competitiveness of male, as evidenced by a mating competitiveness index of 0.17 in laboratory and 0.096 in the orchard. This decline in mating competitiveness is likely linked to the down-regulation of genes associated with the recognition of sex pheromones, specifically CpomOR3a , CpomOR3b , and CpomOR5 , following X-ray irradiation. Fumigation of the plant volatile, linalool at varying concentrations (70, 83, and 96 µL/m³) resulted in differential enhancements in male mating competitiveness, with the moderate concentration significantly improving the competitiveness of sterilized males, possibly by restoring their ability to recognize sex pheromones. Implementation of repeated releases of sterilized males on a pilot scale led to a notable reduction in the population of C. pomonella in the field. These findings indicate that fumigation with plant volatiles has the potential to mitigate male sterility induced by X-ray irradiation, offering a promising approach to enhance the efficacy of SIT applications for the control of C. pomonella . Sterile insect technique Codling moth X-ray Iirradiation Mating competitiveness Plant volatile Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The codling moth, Cydia pomonella (Linnaeus), is a notorious pest that infests pome fruits and walnuts worldwide (Martina et al., 2020 ). Various methods, including agricultural, mechanical, chemical, and biological controls, have been employed to manage the population of C. pomonella (Witzgall et al., 2008 ). Despite efforts to utilize sustainable practices such as agricultural techniques, pheromone-based mating disruption, and attract-kill strategies, effectively controlling dense populations of this pest remains a challenge (Witzgall et al., 2008 ). Traditionally, the predominant approach to combat C. pomonella has been through the use of conventional insecticides (Hu et al., 2020 ). However, the efficacy of chemical insecticides in controlling this pest is limited due to factors like overlapping generations and the larvae's habit of tunneling into fruit (Huang et al., 2024 ; Ju et al., 2021 ). Furthermore, the widespread application of insecticides has led to the development of resistance in C. pomonella (Ju et al., 2021 ). Insect sterility technique (SIT) is recognized as an eco-friendly, efficient, and sustainable approach incorporated within the broader framework of Integrated Pest Management (IPM) (Dyck et al., 2005). The utilization of irradiated males in conjunction with non-irradiated females results in sterile offspring (North, 1975 ). However, the slower progress in developing sterile insect control methods for Lepidoptera in comparison to Diptera is linked to the former's resistance to radiation (Proverbs and Newton, 1962 ). This resistance in Lepidopteran insects is likely due to their necessity for higher radiation doses for sterilization, which can compromise their mating competitiveness and impede the success of sterile insect initiatives. Nevertheless, the effective implementation of the Okanagan-Kootenay Sterile Insect Release (OK SIR) program in British Columbia, Canada led to the successful eradication of C. pomonella populations by strategically employing conventional cobalt-60 ( 60 Co-γ) source in area-wide pest management activities (Anisimov et al., 1989 ). Ongoing endeavors in insect SIT focus on assessing the effects of radiation on insect fertility, longevity, flight capacity, and mating competitiveness (Yamada et al., 2014 ), with the aim of establishing standardized procedures for consistent and reproducible induction of sterility (Yamada et al., 2019 ). The performance of irradiated insects, particularly their mating competitiveness and flight capacity, is of significant importance in the SIT program, as it directly impacts the dispersal range and mating effectiveness of sterile insects in natural environments (Caceres et al., 2007). Previous research has indicated that male pupae irradiated with X-rays at a dose of 366 Gy resulted in complete sterility in C. pomonella , albeit with a low mating competitiveness index of 0.01 (Zhang et al., 2023 ). The observed side effects of X-ray irradiation, such as reduced mating competitiveness, are likely attributed to its negative impact on flight ability (Huang et al., 2024 ). Consequently, it is imperative to optimize irradiation doses and devise strategies to improve both mating competitiveness and flight capacity of sterile insects to ensure the effective implementation of the SIT program for pest management. Host plant volatiles have been documented as influential factors in various behaviors exhibited by phytophagous insects, encompassing mating, feeding, fertilization, directed flight, and pheromone production (Landolt and Philips, 1997). Linalool, a volatile compound categorized as a light-flavored terpene alcohol (Guedes et al., 2004 ), has exhibited a significant attraction towards adult C. pomonella , leading to a considerable electrophysiological reaction from their antennae (Casado et al., 2006 ). Studies conducted on Ceratitis capitata have shown an improvement in mating competitiveness following linalool fumigation, while not impacting the survival rate of sterile males (Guedes et al., 2004 ). Nevertheless, the influence of linalool fumigation on the mating competitiveness of male C. pomonella remains unexplored. The objective of this research is to: 1) ascertain the most effective irradiation dosage to achieve a harmonious equilibrium between sterility and mating competitiveness; 2) develop strategies to enhance the mating competitiveness of sterile insects; 3) elucidate the potential mechanism of the decline in mating competitiveness caused by irradiation and improvement following linalool fumigation; 4) investigate the effect of pilot-release of sterile males on suppression of the C. pomonella field population. 2. Materials and methods 2.1 Insects The C. pomonella strain utilized in the investigation was initially obtained from the Laboratory of Agricultural Invasive Biological Prevention and Monitoring at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. This strain was reared for more than 70 generations within a growth chamber (MLR-352H-PC; Panasonic, Osaka, Japan) under controlled conditions devoid of irradiation. The rearing was maintained a temperature of 26 ± 1°C, relative humidity ranging from 60%±5%, and a photoperiod set at 16 hours of light followed by 8 hours of darkness (Wang et al., 2019). 2.2 Irradiation An X-ray irradiator (SR 100, Changzhou Sairui Instrument Technology Co., Ltd, Changzhou, China) that was furnished with a Radcal Accu-Dose + digitizer featuring a 10 × 6–0.6 CT ion chamber for the purpose of measuring the average dose rate, was utilized in this study. This instrument was employed to expose 8-day-old male pupae (1 day prior to emergence) (Zhang et al., 2023 ), which were positioned within a cylindrical transparent tube (diameter: 2 cm; length: 6 cm) located in the irradiation chamber (dimensions: length: 21 cm; width: 24 cm; height: 16–40 cm) to X-ray radiation at a dose rate of 4.3 Gy/min. 2.3 Effects of different doses of X-ray irradiation on the mating and reproduction of C. pomonella The 8-day-old male pupae were divided into five groups comprising 90 individuals each, and subjected to X-ray irradiation at doses of 0, 50, 100, 150, 200, 250, and 300 Gy, respectively. Subsequently, the pupae within each treatment were randomly divided into three groups consisting of 30 pupae each, and placed in an incubator for emergency situations, with the subsequent emergence count being documented. The matured males and females from the same day were introduced into a mating box (25*12*15 cm) at a ratio of 10 females to 10 males, and each treatment was repeated seven times. Following the laying of eggs by the females and subsequent collection of the egg papers, they were placed in individual disposable transparent plastic bags (15*26 cm) alongside damp wipes on a daily basis, and then transferred to the incubator for hatching. The eggs laid was recorded and the hatching rate was calculated upon the demise of all females. The mortality of the adult was monitored per day, with deceased individuals promptly removed. The lifespan of males was documented, and expired females were gathered in centrifuge tubes (1.5 mL) for dissection under a dissecting microscope to observe mating sac morphology, define mating status, and calculate female mating rates according to Zhang et al. ( 2023 ). Different proportions of "irradiated male: non-irradiated male: non-irradiated female" at ratios of 0:10:10, 10:0:10, and 10:10:10 were established, respectively, to calculate the mating competition index. These male individuals, both irradiated and non-irradiated, along with the female counterparts, were matched in mating age(25*12*15 cm). The eggs laid (fecundity) and hatched (fertility) were meticulously documented for each grouping, with every treatment being replicated thrice for statistical robustness. The competitive mating index (C) of sterile males was then determined utilizing the methodology outlined by Fried ( 1971 ). N is the number of normal male moths and S is the number of irradiated male moths. Ha is the hatching rate of normal male moths paired with normal female moths. Hs is the hatching rate of sterile male moths paired with normal female moths. Ee is the hatching rate of mixed male moths of a certain S/N ratio paired with normal female moths. E is the expected hatching rate of a mixed S/N ratio male moth paired with a normal female moth. 2.4 Effect of linalool fumigation on mating competitiveness of C. pomonella Three disposable transparent plastic boxes (21*14*7 cm) with a volume of 1500 mL each were assembled for experimentation. A strip of filter paper (1 cm in wide and 5 cm long) was suspended in the center of the lid of each box. Male moths that newly emerged were introduced into the boxes. Subsequently, linalool was evenly distributed onto the filter paper strips within the boxes to achieve concentrations of 70, 83, and 96 µL/m³, which were clearly labeled on the lids of the respective boxes. After a 3-hour fumigation period, the moths were removed and the males and females from the same day were segregated into adult mating boxes at a 1:1 ratio. The assessment procedures remained consistent with those described in section 2.3 . 2.5 Effect of linalool fumigation on mating competitiveness of sterile C. pomonella To investigate the effect of linalool fumigation on mating competitiveness of sterile C. pomonella in laboratory, the 8-day-old male pupae were irradiated with X-rays at a dose of 200 Gy, subsequently maintained in a growth chamber until emergence. The freshly emerged male adults underwent a 3-hour fumigation with linalool. The procedures for fumigation, assessment of average survival time, fecundity per female, egg hatchability, mating frequency, and mating competitiveness followed the protocols described above-mentioned. To investigate the effect of linalool fumigation on mating competitiveness of sterile C. pomonella in orchard, a field trial was conducted at Wumeifang Village, Jiuzhai Town, Gaizhou City, Yingkou City, Liaoning Province (40°3′52 "N, 122°5′44 "E) from August 1 to September 2, 2023. Small insect rearing nets (60 cm*60 cm*90 cm) with 40 mesh were affixed to tree trunks, and adult mating age were placed inside these nets for fumigation purposes. The experimental procedures for evaluating mean longevity, single female fecundity, hatching rate, mating rate, and mating competitiveness were consistent with those described above. 2.6 Effects of linalool fumigation on the flight ability of sterile C. pomonella moths The flight performance of moths was assessed using the FXMD-24-USB insect flight information system (Henan Hebi Jiaduoke Industry and Trade Co., Ltd, Hebi, Henan, China), according to the method described in Huang et al. ( 2024 ). Normal, sterile, and fumigated sterile male moths were segregated into distinct adult rearing boxes and provided with a daily diet of 10% honey water. Then, 25 healthy male moths with intact wings that were 3 days post-emergence were selected for the study, and the flight capability of each group was evaluated. The insects were initially immobilized on a chilled surface before their abdomens were exposed. A copper wire was fashioned into a loop, folded at a 90° angle, and affixed to the moth's abdomen using a small amount of adhesive, ensuring proper fixation by inflation and rapid solidification of the glue. The other end of the wire was secured to the arm of the flight mill, positioning the moth parallel to the ground and aligning its flight direction with the mill. Subsequently, the insect flight information system was activated to input data on the test subjects. Flight ability assessments were conducted from 22:00 to 6:00 in a darkened environment, maintaining a constant ambient temperature of 25 ± 1℃ and relative humidity of 70 ± 5% throughout the evaluation process. 2.7 RNA isolation and cDNA synthesis Total RNA was isolated from a collective of ten 3-day-old male adults of C. pomonell a, who were subjected to either 200 Gy X-ray irradiation or not, utilizing the EAStep Super Total RNA Extraction Kit (Promega, Shanghai, China) in accordance with the manufacturer's guidelines. The concentration and quality of the RNA samples were assessed employing a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, 1 µg of RNA from each treatment was utilized in the synthesis of complementary DNA (cDNA) using the GoScript™ Reverse Transcription Mix (Promega, Madison, WI, USA). The resulting cDNA was preserved at -20°C until further analysis. 2.8 Reverse-transcription quantitative polymerase chain reaction analysis The mRNA levels of pheromone recognition-related genes (Table S1 , Figure S1 -S8) were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR) with Bio-Rad CFX96 (Bio-Rad, Hercules, CA, USA) in a reaction of 20 µL, containing of 1 µL of cDNA template, 10 µL of TB Green Premix Ex Taq 2 (TliRNaseH Plus) (TaKaRa, Dalian, China), 1 µL of each primer (10 µmol/L), and 7 µL double-distilled water (ddH2O). The RT-qPCR cycled at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Afterward, a melting curve was conducted starting at 55–95°C to identify the specificity of PCR products. The internal control genes used were EF-1α (MN037793) and β-Actin (KC832921) of C. pomonella (Wei et al., 2020 ). For each three biological replicates and three technical replicates were conducted. Relative expression levels for each gene were calculated using the 2 −△△CT method (Livak and Schmittgen, 2001 ). 2.9 Suppression of field population of C. pomonella by pilot release of sterile males Pilot release of sterile males were conducted between July 8 and September 2, 2023, in Wumeifang Village, Jiuzhai Town, Gaizhou City, Yingkou City, Liaoning Province, China (40°3′52 "N, 122°5′44 "E). Prior to release, a central tree within the orchard was designated and marked, with sex traps positioned at 5-meter intervals in the cardinal directions of east, south, west, and north from the central tree. Sterile males were gently coated with fluorescent powder, distinguishing fumigated and unfumigated specimens by color before being released at 18:00. The control orchard, devoid of sterile male releases, was equipped with 5 trap sets, with trapped moth counts recorded at 10-day intervals post-release. Additionally, daily temperature and precipitation data were collected throughout the release period. The five-point sampling method was employed in both the release orchard and control orchard to designate five points within the central and perimeter areas of the orchard. Around each of these points, five trees were meticulously selected and distinctly labeled. Prior to the release, a comprehensive examination of the total fruit count and the specific number of fruits was conducted. Subsequently, at intervals of 10 days, the quantity of infested fruits was meticulously documented, allowing for the calculation of the fruit infestation rate. This rate was determined by dividing the total number of infested fruits by the overall number of fruits examined, expressed as a percentage. 2.10 Data analysis The statistical significance of various biological parameters of C. pomonella , encompassing metrics such as average emergence rate, longevity, fecundity, sterility rate, mating rate, as well as the flight characteristics of males including flight time, distance, and speed, along with gene expression in controlled laboratory settings and dispersal distance within the orchard, were meticulously analyzed utilizing the one-way analysis of variance (ANOVA) coupled with Duncan's test ( P ≤ 0.05) through SPSS 19 software (IBM Corporation, Armonk, NY, USA). Furthermore, significant difference in total number of moths trapped per trap, amount of insects trapped and fruit decay rate was analyzed through ANOVA with independent samples t-tests (* P < 0.05; ** P < 0.01; *** P < 0.001) using SPSS 19. The results were graphically represented using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA), with all values being expressed as mean ± standard error. Additionally, the sterility rate of C. pomonella under varying X-ray dosage levels was modeled using a logistic curve in Origin 2019 software (Origin Lab Corporation, Northampton, MA 02115, USA). 3. Results 3.1 Effect of different doses of X-ray irradiation on the biological parameters of C. pomonella The effects of different doses of X-ray irradiation on the physiological traits of C. pomonella were explored in a series of experiments that examined the emergence rate, lifespan, reproductive capacity, and sterility of 8-day-old pupae exposed to varying levels of X-ray radiation. The results indicated that there was no statistically significant variance in the emergence rate of male moths C. pomonella within the range of 50–250 Gy compared to the control group. Notably, a reduction in the emergence rate of male C. pomonella moths was evident at 300 Gy (Fig. 1 a). However, the survival rate of moths arising from male pupae irradiated with various doses of radiation did not exhibit a significant difference compared to the control group (Fig. 1 b). Furthermore, a decrease in lifespan was evident with X-ray exposure surpassing 200 Gy when compared to the control group (Fig. 1 c). Fecundity levels remained steady across different radiation levels compared to the control group (Fig. 1 d). Additionally, an escalation in sterility rate was observed at doses below 250 Gy (Fig. 1 e), while a reduction in mating rate was noted at doses exceeding 200 Gy (Fig. 1 f). 3.2 Effect of 200 Gy X-ray irradiation on mating competitiveness of male 3. 2 Effect of 200 Gy X-ray irradiation on mating competitiveness of male C. pomonella 3.2.1 Laboratory test The hatching rate of eggs for SM:NM:NF at the 1:0:1 ratio was found to be 12.57 ± 0.51%, a value significantly lower compared to the ratios of 0:1:1 (72.96 ± 7.31%) and 1:1:1 (64.18 ± 3.95%). When male adults were combined with different ratios and matched with normal female adults, the expected hatching rate (E) was 42.77%. The competitive mating index (C) was determined to be 0.17, indicating that the mating competitiveness of male moths was reduced by 200 Gy of irradiation (Table 1 ). Table 1 200Gy X-ray irradiation on the mating competitiveness of C. pomonella adults Experimental site Matching ratio (S♂: N♂: N♀) Fecundity (eggs laid per female) Hatching rate/% Competitive capacity Observed value Expected value Indoor experiment 0 : 10 : 10 116.20 ± 19.97 a 72.96 ± 7.31 a 10 : 0 : 10 103.75 ± 4.42 a 12.57 ± 0.51 b 10 : 10 : 10 106.07 ± 5.80 a 64.18 ± 3.95 a 42.77 0.17 Field experiment 0 : 10 : 10 57.45 ± 4.09 a 64.16 ± 6.11 a 10 : 0 : 10 60.13 ± 10.60 a 10.82 ± 4.46 b 10 : 10 : 10 54.26 ± 6.81 a 59.49 ± 2.47 a 37.49 0.096 Data are mean ± SE. N: Number of unirradiated males; S: Number of irradiated males. 3.2.2 Orchard trial The hatching rate of eggs for the SM:NM:NF group at the 1:0:1 ratio was observed to be 10.82 ± 4.46%, a significantly lower rate compared to the 0:1:1 ratio (64.16 ± 6.11%) and the 1:1:1 ratio (59.49 ± 2.47%). When male adults were introduced in various ratios and paired with normal female adults, the expected hatching value (E) was calculated to be 37.49%. Additionally, the competitive mating index (C) was determined to be 0.096, suggesting that a radiation dosage of 200 Gy diminished the mating competitiveness of male moths (Table 1 ). 3.3 Effects of different concentrations of linalool fumigation on biological parameters of C. pomonella To elucidate the impact of varying concentrations of linalool fumigation treatments on the biological parameters of C. pomonella , experimental investigations were conducted to assess the survival duration, oviposition, egg hatching efficacy, and mating activity of male moths. The results revealed no notable disparities in the aforementioned parameters among male moths subjected to distinct levels of linalool fumigation in comparison to the control cohort, indicating that the application of linalool fumigation did not adversely affect the male moths (Fig. 2 ). 3.4 Effects of different concentrations of linalool fumigation on mating competitiveness of C. pomonella The hatching rate of eggs resulting from the pairing of fumigation-treated normal males with normal females were determined to be 73.63 ± 3.86 percent, while the pairing of untreated normal males with normal females yielded a hatching rate of 71.42 ± 1.74%. When comparing the hatching rate of fumigation-treated normal males to the overall hatching rate, the value was found to be 72.55 ± 0.33%. There was no significant difference observed in the hatchability of fecundity across three different mating combinations, with an average rate of 72.52%. Furthermore, the mating competitiveness index of C. pomonella normal males that were exposed to linalool fumigation at a concentration of 70µL/m³was calculated to be 1.06, suggesting an enhancement in the mating competitiveness of the males (Table 2 ). Table 2 Effect of different concentrations of linalool fumigation on the mating competitiveness of C. pomonella males Fumigation concentration (µL/m³ ) Matching ratio (F♂: N♂: N♀) Fecundity (eggs laid per female) Hatching rate/% Competitive capacity Observed value Expected value 70 0 : 10 : 10 95.41 ± 1.98 a 71.42 ± 1.74 a 10 : 0 : 10 96.46 ± 9.51 a 73.63 ± 3.86 a 10 : 10 : 10 98.74 ± 9.33 a 72.55 ± 0.33 a 72.52 1.06 83 0 : 10 : 10 95.41 ± 1.98 a 71.42 ± 1.74 a 10 : 0 : 10 94.75 ± 13.79 a 78.47 ± 3.28 a 10 : 10 : 10 95.83 ± 2.05 a 75.34 ± 0.43 a 74.94 1.25 96 0 : 10 : 10 95.41 ± 1.98 a 71.42 ± 1.74 a 10 : 0 : 10 89.55 ± 3.98 a 76.52 ± 1.36 a 10 : 10 : 10 95.02 ± 0.89 a 74.24 ± 3.83 a 73.97 1.24 The percentage of eggs hatched when 83 µL/m³ of linalool was used to fumigate normal males paired with normal females was recorded at 78.47 ± 3.28%. For normal males paired with normal females, the hatching rate was slightly lower at 71.42 ± 1.74%. When fumigated normal males were paired with normal females, the hatching rate was 75.34 ± 0.43%. There was no significant difference in the hatching rate of eggs produced by the three different mating combinations, all averaging around the expected value of 74.94. The mating competitiveness index of males treated with linalool at 83 µL/m³was 1.25, indicating an improvement in mating competitiveness compared to those treated at 70 µL/m³ (Table 2 ). The egg hatching rate of 96 µL/m³ linalool-exposed normal male moths when paired with normal females was determined to be 76.52 ± 1.36%, while that of normal males paired with normal females was 71.42 ± 1.74%, and the hatching rate of fumigated normal males when paired with other males and females was 74.24 ± 3.83%. These results indicate that there were no significant differences in hatching rates among the three mating combinations studied, all of which were close to the anticipated value of 73.97%. Furthermore, the mating competitiveness index of males exposed to linalool at 96µL/m³was calculated to be 1.24, suggesting a reduced mating competitiveness compared to males exposed to linalool at 83 µL/m³ (Table 2 ). 3.5 Effect of linalool fumigation on mating competitiveness of sterilized male C. pomonella moths 3.5.1 Laboratory test The results revealed that male moths exposed to 200 Gy X-rays exhibited notably reduced hatching and mating frequencies in comparison to the untreated group. Notably, there were no substantial variations in any of the outcomes between the fumigated and non-fumigated sterilized males, indicating that the linalool fumigation did not impact the reproductive characteristics of sterilized males (Fig. 3 ). Specifically, the hatching rate of linalool-fumigated irradiated sterilized males that were paired with normal females was 13.44 ± 2.33%, while that of irradiated sterilized males paired with normal females stood at 10.97 ± 2.41%. Moreover, the hatching rate of fumigation-treated irradiated sterilized males when paired with females was 12.27 ± 1.98%, suggesting no significant variance in the hatching rate of eggs produced by the three different pairings (F = 0.305, df = 8, P = 0.748), with an expected value of 12.21%. The mating competition index of 1.11 for the irradiated sterilized males of moths fumigated with linalool indicated an enhancement in the mating competitiveness of the irradiated sterilized males due to linalool fumigation (Table 3 ). Table 3 Effect of different concentrations of linalool fumigation on the mating competitiveness of 200 Gy X-ray sterilized C. pomonella males Experimental site Matching ratio (F♂: N♂: N♀) Fecundity(eggs laid per female) Hatching rate/% Competitive capacity Observed value Expected value Indoor experiment 0 : 10 : 10 106.54 ± 5.71 a 10.97 ± 2.41 a 10 : 0 : 10 103.30 ± 12.00 a 13.44 ± 2.33 a 10 : 10 : 10 94.35 ± 11.43 a 12.27 ± 1.98 a 12.21 1.11 Field experiment 0 : 10 : 10 60.13 ± 10.60 a 10.82 ± 4.46 a 10 : 0 : 10 55.40 ± 5.28 a 14.98 ± 3.89a 10 : 10 : 10 61.66 ± 4.09 a 12.84 ± 1.98 a 12.90 1.06 3.5.2 Orchard trial The results of the field experiment revealed a significant decrease in the longevity and fecundity of sterile males of C. pomonella compared to the control group. Interestingly, there was no notable variance between the biological parameters of linalool-fumigated sterile males and non-fumigated ones, suggesting that the fumigation treatments did not impact these parameters within the field environment (Fig. 3 ). When linalool-fumigated irradiated sterile males were paired with normal females, the hatchability was observed to be 14.98 ± 3.89%, while it was 10.82 ± 4.46% for C. pomonella irradiated sterile males mated with normal females. Moreover, the hatchability of fumigation-treated irradiated sterile males when paired with females stood at 12.84 ± 1.98%. These findings indicate that there was no significant difference in hatchability and fecundity among the three different combinations (F = 0.249, df = 8, P = 0.756), with an average value of 12.90%. The mating competition index of 1.06 for the linalool-fumigated irradiated sterile males of C. pomonella suggests that linalool fumigation enhanced the mating competitiveness of the irradiated sterile males (Table 3 ). 3.6 Effects of linalool fumigation on the flight ability of sterile C. pomonella moths The flight duration of 3-day-old male adults, sterile males, and fumigation treated males was 53.82 ± 7.17, 5.61 ± 2.36, and 10.18 ± 3.46 min, respectively. While no significant disparity was observed in the flight duration between sterile males and fumigation treated males, both groups exhibited significantly shorter flight times compared to the control group (F = 30.820, df = 44, P < 0.001). Moreover, there were no significant differences in flight distance or flight speed among the treatment groups when compared to the control group, (Fig. 5 ). 3.7 Effects of linalool fumigation on the expression levels of sex pheromone receptor-related genes in sterile C. pomonella moths RT-qPCR results showed that CpomOR1, CpomOR2a, CpomOR3a, CpomOR3b, CpomOR5, CpomOR6a, CpomOR7, CpomGOBP1 , and CpomGOBP2 genes were significantly reduced in C. pomonella male moths that emerged from pupae irradiated with 200 Gy X-ray (Fig. 6 ). The expression levels of CpomOR3a, CpomOR3b , and CpomOR5 were significantly rebounded after linalool fumigation (Fig. 6 ). 3.8 Suppression of field population of C. pomonella by pilot release of sterile males A total of 5342 sterile males were released, with 2735 of them being released from linalool fumigated moths. Among these, 60.44 ± 4.48% of males flew out normally, while 6.83 ± 1.51% fell to the ground post take-off, leaving 39.56 ± 4.48% unable to take off normally (Table S2). The percentage of males falling to the ground was 59.84 ± 6.86%. Additionally, 2607 sterile males of C. pomonella without linalool fumigation were also released, where 62.17 ± 4.25% flew out normally, 17.07 ± 2.86% fell to the ground after take-off, and 37.83 ± 4.25% couldn't take off normally. Notably, 78.19 ± 6.36% of males picked with tweezers failed to take off and fell to the ground (Table S3). Throughout the experiment, the ambient temperature at the experimental site exhibited minimal variations, with a singular precipitation event exceeding 100 mm (Fig. 7 a). Results revealed that, at a 5-meter distance, the recapture of sterile male moths in traps averaged 12.25 ± 3.07, whereas the recapture of fumigation-treated sterile males was 8.75 ± 3.16. Similarly, at distances of 10 m, the recaptures were 10.00 ± 1.78 and 9.75 ± 1.18, respectively. At 15 m, the retrievals were 8.50 ± 1.55 and 6.50 ± 1.19, respectively. Finally, at a distance of 20 m, the retrievals were 0 ± 0 and 1.50 ± 0.65, respectively. The results indicated that there were no statistically significant variations in the recaptures of sterile males and fumigation-treated sterile males of C. pomonella at the 10 and 20 m distances at a significance level of 0.05 (P > 0.05). However, notable differences were noted in recaptures at 5 and 15 meters, with statistically significant variances observed at the 0.05 level (P < 0.05) (Fig. 7 b). The deployment of C. pomonella sterile males over a period of two months resulted in a notable decline in fruit infestation within the treated orchards, registering at 24.87 ± 2.17%. This rate was significantly lower ( P < 0.05) in contrast to the control orchard where infestation levels reached 34.01 ± 2.87%. These findings provide evidence that the introduction of sterile C. pomonella males effectively mitigated fruit infestation occurrences, thereby diminishing both the incidence of infested fruits and the overall infestation rate within the orchard (Fig. 7 C). The captures of wild male moths did not exhibit a notable variance between the release area and the control from July 7 to July 14, 2023. Nonetheless, subsequent to July 31, 2023, a noteworthy decline in captures within the release area was observed, presenting a significant contrast with the control group (P < 0.05). Based on these findings, it is evident that the introduction of sterile male C. pomonella moths in the natural environment substantially reduces the population of wild male moths (Fig. 7 d). 4 Discussion The radiation dose is a critical factor in achieving the desired sterility level of insects in the SIT program (Proverbs and Newton, 1962 ). In this study, we demonstrated that the biological traits of male C. pomonella varied in their decline with increasing levels of irradiation. This observation is consistent with a previous study on Glossina palpalis tsetse flies, which showed that higher radiation doses reduced the fecundity of females (Pagabeleguem et al., 2023 ). Additionally, we observed a significant decrease in the mating rate of sterile male C. pomonella with normal females when exposed to radiation above 200 Gy, mirroring the findings in G. palpalis (Pagabeleguem et al., 2023 ). The OK SIR program, designed to control C. pomonella populations, initially utilized 250 Gy of 60 Co-γ irradiation, resulting in male sterility rates of 85–92% (Holm, 1985 ). Since 2014, a consistent dose of 200 Gy has been employed to balance sterility levels and mating competitiveness (Thistlewood and Judd, 2019 ). To optimize the trade-off between male sterility and minimize the negative effects of radiation on the insects, it is recommended to use 200 Gy as the optimal dose for inducing sterility in males C. pomonella . The mating competitiveness of sterile insects is crucial for their ability to effectively compete with wild males for mating opportunities with females (Vreysen et al., 2007 ; Vreysen and Robinson, 2011 ). Previous research has shown that inadequate dosages may result in incomplete sterilization, while excessive irradiation can reduce the mating competitiveness of released insects compared to wild counterparts, thus affecting the success of SIT programs (Parker et al., 2021 ). Therefore, maintaining a delicate balance between mating competitiveness and sterility is essential during copulatory interactions with wild female insects (Bakri et al., 2005 ). Evaluating the optimal irradiation dose is critical to assessing the mating competitiveness of male insects bred for this purpose (Parker et al., 2021 ). However, reduced mating competitiveness is commonly observed in irradiated C. pomonella populations (North, 1975 ). Jiang et al. ( 2023 ) also reported a significant decrease in the mating competitiveness of male Spodoptera frugiperda following exposure to 250 Gy of X-ray irradiation. In our recent studies, male C. pomonella pupae irradiated with 366 Gy of X-rays exhibited mating competitiveness indices of 0.001 in laboratory (Zhang et al., 2023 a) and 0.0088 in orchards (Zhang et al., 2023 b). In this study, when subjected to 200 Gy of X-ray irradiation, the mating competitiveness index of sterilized male C. pomonella decreased, indicating a decline in mating competitiveness post-irradiation. Nevertheless, compared to the impact of 366 Gy irradiation, the mating competitiveness index of C. pomonella males following 200 Gy irradiation increased to 0.17 in laboratory conditions and 0.096 in orchards, suggesting that an optimized irradiation dose can enhance the mating competitiveness of sterile insects to a certain degree. However, the mating competitiveness of C. pomonella males post-exposure to 200 Gy irradiation persists at a sub-optimal level. Therefore, it is imperative to prioritize efforts aimed at improving the mating competitiveness of sterile males in future studies. The diminished mating competitiveness evident in irradiated insects may stem from the compromised olfactory system in male insects, leading to a reduced ability to perceive female sex pheromones. To investigate this hypothesis, linalool, a volatile compound found in host plants known to enhance the attraction of C. capitata moths (Casado et al., 2006 ), was utilized to fumigate male C. pomonella moths. The findings indicated that although linalool fumigation did not affect longevity, fecundity, hatching rates, or overall mating performance, it did enhance the mating competitiveness of male moths. Another investigation on C. capitata demonstrated that the mating competitiveness of normal male adults and mass-reared irradiated males increased subsequent to fumigation with ginger root oil (GRO), compared to non-fumigated adults. Notably, GRO fumigation did not have any notable impacts on the body weight or longevity of irradiated mass-reared males (Shelly and McInnis, 2001). These results suggest that linalool fumigation might potentially mitigate male sterility induced by X-ray irradiation, possibly due to enhanced male sex pheromone communication in the antennae of male C. pomonella moths, as observed in C. capitata post ginger root oil fumigation (Papadopoulos et al., 2006 ). The olfactory system plays a vital role in various insect behaviors like feeding, orientation, host-seeking, mating, and oviposition (Li et al., 2020 ; Zhang et al., 2019 ). Chemoreception in insects is a complex process involving various chemosensory genes, such as odorant receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs), odorant-binding proteins (OBPs), chemosensory proteins (CSPs), and sensory neuron membrane proteins (SNMPs) (Yang et al., 2024 ). In particular, the importance of chemosensory genes in recognizing pheromones in C. pomonella has been extensively researched. Tian et al. ( 2021 ) have identified male pheromone receptor genes in C. pomonella , including CpomOR1 , CpomOR2a , CpomOR3 , CpomOR5 , CpomOR6a , and CpomOR7 , through transcriptome analysis of the antenna. Knock-out the CpomOR1 gene using CRISPR/Cas9 technology has been shown to impact the fertility and fecundity of C. pomonella (Garczynski et al., 2017 ). Moreover, CpomOR3a and CpomOR3b genes in C. pomonella are involved in recognizing plant volatiles and the sex pheromone codlemone, thereby enhancing the moths' response to codlemone (Wan et al., 2019 ). In this study, we revealed a decrease in gene expression in C. pomonella males' antenna following 200 Gy of X-ray irradiation, suggesting a reduced sex pheromones response in male moths. Conversely, an increase in the expression of CpomOR3a , CpomOR3b , and CpomOR5 genes was observed after linalool fumigation, enhancing the recognition of sex pheromones.These findings support the efficacy of linalool fumigation in mitigating male sterility induced by X-ray irradiation, likely through the upregulation of essential chemosensory genes and the activation of pheromone recognition mechanisms in C. pomonella . Previous studies have demonstrated that the release of C. pomonella males into the pear orchard following exposure to 366 Gy X-ray did not affect the wild populations or the fruit decay rate (Zhang et al., 2023 ). In this study, we carried out a small-scale trial in different fields utilizing the SI) to manage C. pomonella populations. Our study demonstrated that by releasing three successive batches of sterile male C. pomonella moths irradiated at 200 Gy, along with linalool fumigation, in the specified orchard area, there was a substantial decrease in C. pomonella infestations in the apple crop. This reduction was evident through significantly lower infestation rates compared to the control orchard. Additionally, field observations indicated a notable decrease in the recapture rate of wild C. pomonella moths in comparison to the control group. These results suggest that the utilization of sterile males effectively suppressed the wild C. pomonella population in the orchards, consequently reducing fruit damage. The findings highlight an enhancement in the field application of the SIT for C. pomonella control after optimizing the irradiation dose. In summary, we have identified the optimal sterilizing dose of X-ray irradiation for C. pomonella and introduced a plant volatiles fumigation approach to enhance the mating competitiveness of sterilized males. Additionally, we investigated the effects of irradiation and fumigation on the expression of genes related to pheromone recognition in the male antennal. Notably, our study demonstrated the successful reduction of the C. pomonella population in the orchards through repeated releases of sterilized males in conjunction with linalool fumigation on a pilot scale. These findings offer valuable insights for the implementation of the SIT as a strategy of controlling C. pomonella . Declarations Author Contribution Sheng-Wang Huang: software, methodology, investigation, formal analysis, writing original draft, Peng-Cheng Wang: software, methodology, investigation, formal analysisYan Wang: software, review and editing. Jie-Qiong: formal analysis, conceptualization. Ping Gao: writing, review, and editing, resources.Xue-Qing Yang: supervision, resources, project administration, funding acquisition, conceptualization, writing, review, and editing. Acknowledgement This research was funded by the National Key R&D Program of China (2021YFD1400200, 2023YFD1400704) and the IAEA TC program (CPR5027). References Anisimov AI, Lazurkina NV, and Shvedov AN (1989) Influence of radiation-induced genetic damage on the suppressive effect of inherited sterility in the codling moth (Lepidoptera: Tortricidae). Ann. Entomol. Soc. Am. 82: 769–777. https://doi.org/10.1093/aesa/82.6.769 Bakri A., Mehta K., and Lance DR. (2005) Sterilizing insects with radiation. 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Zhang J, Huang S, Zhao S, et al (2023) The effect of X-ray irradiation on the fitness and field adaptability of the codling moth: an orchard study in Northeast China. Insects 14(7): 615. https://doi.org/10.3390/insects14070615 Additional Declarations No competing interests reported. Supplementary Files Supportinginformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4677671","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":331659906,"identity":"61e8be16-d264-4679-a443-1e07e56cb893","order_by":0,"name":"Sheng-Wang Huang","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Sheng-Wang","middleName":"","lastName":"Huang","suffix":""},{"id":331659908,"identity":"84927d16-f2f1-4dc5-a8c9-1ccd5be5f68c","order_by":1,"name":"Peng-Cheng Wang","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Peng-Cheng","middleName":"","lastName":"Wang","suffix":""},{"id":331659910,"identity":"4f39a092-aadb-4728-b790-071f5e2eb09a","order_by":2,"name":"Yan Wang","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Wang","suffix":""},{"id":331659912,"identity":"5fac6f0c-a676-42fe-8fa2-47aaf1127d04","order_by":3,"name":"Jie-Qiong Wang","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jie-Qiong","middleName":"","lastName":"Wang","suffix":""},{"id":331659913,"identity":"f478f3c3-855a-44fb-8be5-519f467093bb","order_by":4,"name":"Ping Gao","email":"","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Gao","suffix":""},{"id":331659915,"identity":"333d2bb4-56f7-4b6c-bfa7-91b5eeb95628","order_by":5,"name":"Xue-Qing Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACdsYGBoaKGjt+CTBXQoawFmaQljPHkiVnMIBYEjxEaAFixjZmxg03wFoYCGuRb2Zue/iFjY3Z+Hbz8Uc3aix4GNgPH92AT4vBYcZ2YxkeGT6zO8cSm3OOAR3Gk5Z2A68WZsY2aQkJNmazGzmGzTlsQC0SPGZ4tcg3g7QANW6eAdLyjwgtDIcZ2yQ/JAC9LwHUkttGhBagX9qkGQ4cS5a4kZY4O7dPgoeNkF/k29ufSf78B4zKGckHPud8q5PjZz98DL/DgIAZJS7YCCkHAcYfxKgaBaNgFIyCkQsA54NC0zdySLQAAAAASUVORK5CYII=","orcid":"","institution":"Shenyang Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xue-Qing","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-07-03 05:08:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4677671/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4677671/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61346554,"identity":"dd3eea21-6824-4651-8d0f-e78e5d1bbf08","added_by":"auto","created_at":"2024-07-29 18:08:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":664351,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different doses of X-ray irradiation on the biological parameters of male \u003cem\u003e\u003cstrong\u003eC. pomonella\u003c/strong\u003e\u003c/em\u003e. (a) Emergence rate; (b) Survival rate; (c) Longevity; (d) Fecundity (eggs laid per female); (e) Sterility rate; (f) Mating rate. Letters on the error bars indicate significant differences analyzed by the one-way analysis of variance (ANOVA) with Duncan’s test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4677671/v1/b9b650b7f03eb5101e42192f.png"},{"id":61346553,"identity":"f136578b-999c-4599-8f1b-5e39301ec305","added_by":"auto","created_at":"2024-07-29 18:08:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":437436,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different concentrations of linalool fumigation on the biological parameters of male \u003cem\u003e\u003cstrong\u003eC. pomonella\u003c/strong\u003e\u003c/em\u003e. (a) Survival rate; (b) Longevity; (c) Fecundity (eggs laid per female); (d) Hatching rate; (e) Mating rate.Letters on the error bars indicate significant differences analyzed by the one-way analysis of variance (ANOVA) with Duncan’s test.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4677671/v1/520dc16915fb15be3043c2e2.png"},{"id":61346557,"identity":"1efda62d-cef2-4446-9bf6-d277c0b0391c","added_by":"auto","created_at":"2024-07-29 18:08:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":153533,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different concentrations of linalool fumigation on the biological parameters of 200 Gy X-ray sterilized male \u003cem\u003e\u003cstrong\u003eC. pomonella \u003c/strong\u003e\u003c/em\u003e(laboratory experiment). (a) Survival rate; (b) Longevity; (c) Fecundity (eggs laid per female); (d) Hatching rate; (e) Mating rate.Letters on the error bars indicate significant differences analyzed by the one-way analysis of variance (ANOVA) with Duncan’s test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4677671/v1/b99cc3082eacad5e838ef9e1.png"},{"id":61346556,"identity":"cb0b51e9-3e7d-4dfd-ade7-bb33c7c1a5d5","added_by":"auto","created_at":"2024-07-29 18:08:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":133850,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different concentrations of linalool fumigation on the biological parameters of 200 Gy X-ray sterilized male \u003cem\u003e\u003cstrong\u003eC. pomonella \u003c/strong\u003e\u003c/em\u003e(field experiment). (a) Survival rate; (b) Longevity; (c) Fecundity (eggs laid per female); (d) Hatching rate; (e) Mating rate.Letters on the error bars indicate significant differences analyzed by the one-way analysis of variance (ANOVA) with Duncan’s test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4677671/v1/689575b3b603fb4086fc2dcf.png"},{"id":61346558,"identity":"3d60ff21-c7ab-4cba-b237-8dbf329cf933","added_by":"auto","created_at":"2024-07-29 18:08:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81960,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of X-ray irradiation on the flight ability of male adults of \u003cem\u003eC. pomonella. \u003c/em\u003e(a) Flight duration; (b) Flight distance; (c) Average flight speed. Asterisk (*) on the error bars indicate significant differences analyzed by the one-way analysis of variance (ANOVA) with independent samples t-tests (*, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4677671/v1/c8c1d3185b05dcf1fd975c5d.png"},{"id":61346560,"identity":"0addfbc6-9305-4641-a104-8f07b0f1b412","added_by":"auto","created_at":"2024-07-29 18:08:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2590059,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of X-ray irradiation on the relative expression levels of the pheromone recognition-related genes in male adults of\u003cem\u003e C. pomonell\u003c/em\u003ea. (a) \u003cem\u003eCpomOR1\u003c/em\u003e; (b)\u003cem\u003e CpomOR2\u003c/em\u003ea; (c) \u003cem\u003eCpomOR3\u003c/em\u003ea; (d)\u003cem\u003e CpomOR3b\u003c/em\u003e; (e) \u003cem\u003eCpomOR5\u003c/em\u003e; (f)\u003cem\u003e CpomOR6a\u003c/em\u003e; (g) \u003cem\u003eCpomOR\u003c/em\u003e7; (h) \u003cem\u003eCpomGOBP\u003c/em\u003e1; (i) \u003cem\u003eCpomGOBP2\u003c/em\u003e; (j) \u003cem\u003eCpomGOBP3\u003c/em\u003e. The 8-day-old male pupae (1 day before emergence) were exposed to 200 Gy of X-ray, and the expression levels of pheromone receptor-related genes in 3-day-old male adults were analyzed. Letters on the error bars indicate significant differences analyzed by the one-way analysis of variance (ANOVA) with Duncan’s test.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4677671/v1/171c0e5f24483249a362a494.png"},{"id":61346559,"identity":"d42dbcec-c8b5-44c8-a8c2-13be341d7dbd","added_by":"auto","created_at":"2024-07-29 18:08:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2043604,"visible":true,"origin":"","legend":"\u003cp\u003eSuppression of field population of \u003cem\u003eC. pomonella\u003c/em\u003e by pilot release of sterile male moths in an apple orchard.\u003cstrong\u003e \u003c/strong\u003e(a) Mean air temperature and daily precipitation in the field during the experimental period. (b) The trapping amount of wild male insects in the control and release areas.(c) Survey of fruit decay rate in control and release areas. The red triangles represent the date of release. (d) Number of sterile males recaptured by traps at different distances. (*) on the error bars indicate significant differences analyzed by the one-way analysis of variance (ANOVA) with independent samples t-tests (*, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4677671/v1/aae957cd1d929af04b9ba86e.png"},{"id":61349661,"identity":"a54ce0cf-53a0-4de0-8c86-dd448fc0e06e","added_by":"auto","created_at":"2024-07-29 18:32:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7136015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4677671/v1/30dcd0a0-fd3a-4f4e-954c-e76e36d01b5f.pdf"},{"id":61346561,"identity":"8518bec7-11d1-49d6-84b1-55126d33ce16","added_by":"auto","created_at":"2024-07-29 18:08:38","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":8438329,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4677671/v1/a0ec15c9c9517fcc3e60ebc3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plant volatile-based fumigation improves mating competitiveness of males for population suppression of the global fruit pest Cydia pomonella","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe codling moth, \u003cem\u003eCydia pomonella\u003c/em\u003e (Linnaeus), is a notorious pest that infests pome fruits and walnuts worldwide (Martina et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Various methods, including agricultural, mechanical, chemical, and biological controls, have been employed to manage the population of \u003cem\u003eC. pomonella\u003c/em\u003e (Witzgall et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Despite efforts to utilize sustainable practices such as agricultural techniques, pheromone-based mating disruption, and attract-kill strategies, effectively controlling dense populations of this pest remains a challenge (Witzgall et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Traditionally, the predominant approach to combat \u003cem\u003eC. pomonella\u003c/em\u003e has been through the use of conventional insecticides (Hu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the efficacy of chemical insecticides in controlling this pest is limited due to factors like overlapping generations and the larvae's habit of tunneling into fruit (Huang et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ju et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, the widespread application of insecticides has led to the development of resistance in \u003cem\u003eC. pomonella\u003c/em\u003e (Ju et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInsect sterility technique (SIT) is recognized as an eco-friendly, efficient, and sustainable approach incorporated within the broader framework of Integrated Pest Management (IPM) (Dyck et al., 2005). The utilization of irradiated males in conjunction with non-irradiated females results in sterile offspring (North, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). However, the slower progress in developing sterile insect control methods for Lepidoptera in comparison to Diptera is linked to the former's resistance to radiation (Proverbs and Newton, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). This resistance in Lepidopteran insects is likely due to their necessity for higher radiation doses for sterilization, which can compromise their mating competitiveness and impede the success of sterile insect initiatives. Nevertheless, the effective implementation of the Okanagan-Kootenay Sterile Insect Release (OK SIR) program in British Columbia, Canada led to the successful eradication of \u003cem\u003eC. pomonella\u003c/em\u003e populations by strategically employing conventional cobalt-60 (\u003csup\u003e60\u003c/sup\u003e Co-γ) source in area-wide pest management activities (Anisimov et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Ongoing endeavors in insect SIT focus on assessing the effects of radiation on insect fertility, longevity, flight capacity, and mating competitiveness (Yamada et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), with the aim of establishing standardized procedures for consistent and reproducible induction of sterility (Yamada et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe performance of irradiated insects, particularly their mating competitiveness and flight capacity, is of significant importance in the SIT program, as it directly impacts the dispersal range and mating effectiveness of sterile insects in natural environments (Caceres et al., 2007). Previous research has indicated that male pupae irradiated with X-rays at a dose of 366 Gy resulted in complete sterility in \u003cem\u003eC. pomonella\u003c/em\u003e, albeit with a low mating competitiveness index of 0.01 (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The observed side effects of X-ray irradiation, such as reduced mating competitiveness, are likely attributed to its negative impact on flight ability (Huang et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, it is imperative to optimize irradiation doses and devise strategies to improve both mating competitiveness and flight capacity of sterile insects to ensure the effective implementation of the SIT program for pest management.\u003c/p\u003e \u003cp\u003eHost plant volatiles have been documented as influential factors in various behaviors exhibited by phytophagous insects, encompassing mating, feeding, fertilization, directed flight, and pheromone production (Landolt and Philips, 1997). Linalool, a volatile compound categorized as a light-flavored terpene alcohol (Guedes et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), has exhibited a significant attraction towards adult \u003cem\u003eC. pomonella\u003c/em\u003e, leading to a considerable electrophysiological reaction from their antennae (Casado et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Studies conducted on \u003cem\u003eCeratitis capitata\u003c/em\u003e have shown an improvement in mating competitiveness following linalool fumigation, while not impacting the survival rate of sterile males (Guedes et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Nevertheless, the influence of linalool fumigation on the mating competitiveness of male \u003cem\u003eC. pomonella\u003c/em\u003e remains unexplored.\u003c/p\u003e \u003cp\u003eThe objective of this research is to: 1) ascertain the most effective irradiation dosage to achieve a harmonious equilibrium between sterility and mating competitiveness; 2) develop strategies to enhance the mating competitiveness of sterile insects; 3) elucidate the potential mechanism of the decline in mating competitiveness caused by irradiation and improvement following linalool fumigation; 4) investigate the effect of pilot-release of sterile males on suppression of the \u003cem\u003eC. pomonella\u003c/em\u003e field population.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Insects\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eC. pomonella\u003c/em\u003e strain utilized in the investigation was initially obtained from the Laboratory of Agricultural Invasive Biological Prevention and Monitoring at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. This strain was reared for more than 70 generations within a growth chamber (MLR-352H-PC; Panasonic, Osaka, Japan) under controlled conditions devoid of irradiation. The rearing was maintained a temperature of 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, relative humidity ranging from 60%\u0026plusmn;5%, and a photoperiod set at 16 hours of light followed by 8 hours of darkness (Wang et al., 2019).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Irradiation\u003c/h2\u003e \u003cp\u003eAn X-ray irradiator (SR 100, Changzhou Sairui Instrument Technology Co., Ltd, Changzhou, China) that was furnished with a Radcal Accu-Dose\u0026thinsp;+\u0026thinsp;digitizer featuring a 10 \u0026times; 6\u0026ndash;0.6 CT ion chamber for the purpose of measuring the average dose rate, was utilized in this study. This instrument was employed to expose 8-day-old male pupae (1 day prior to emergence) (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which were positioned within a cylindrical transparent tube (diameter: 2 cm; length: 6 cm) located in the irradiation chamber (dimensions: length: 21 cm; width: 24 cm; height: 16\u0026ndash;40 cm) to X-ray radiation at a dose rate of 4.3 Gy/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Effects of different doses of X-ray irradiation on the mating and reproduction of \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe 8-day-old male pupae were divided into five groups comprising 90 individuals each, and subjected to X-ray irradiation at doses of 0, 50, 100, 150, 200, 250, and 300 Gy, respectively. Subsequently, the pupae within each treatment were randomly divided into three groups consisting of 30 pupae each, and placed in an incubator for emergency situations, with the subsequent emergence count being documented. The matured males and females from the same day were introduced into a mating box (25*12*15 cm) at a ratio of 10 females to 10 males, and each treatment was repeated seven times. Following the laying of eggs by the females and subsequent collection of the egg papers, they were placed in individual disposable transparent plastic bags (15*26 cm) alongside damp wipes on a daily basis, and then transferred to the incubator for hatching. The eggs laid was recorded and the hatching rate was calculated upon the demise of all females. The mortality of the adult was monitored per day, with deceased individuals promptly removed. The lifespan of males was documented, and expired females were gathered in centrifuge tubes (1.5 mL) for dissection under a dissecting microscope to observe mating sac morphology, define mating status, and calculate female mating rates according to Zhang et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDifferent proportions of \"irradiated male: non-irradiated male: non-irradiated female\" at ratios of 0:10:10, 10:0:10, and 10:10:10 were established, respectively, to calculate the mating competition index. These male individuals, both irradiated and non-irradiated, along with the female counterparts, were matched in mating age(25*12*15 cm). The eggs laid (fecundity) and hatched (fertility) were meticulously documented for each grouping, with every treatment being replicated thrice for statistical robustness. The competitive mating index (C) of sterile males was then determined utilizing the methodology outlined by Fried (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1971\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e is the number of normal male moths and \u003cem\u003eS\u003c/em\u003e is the number of irradiated male moths. \u003cem\u003eHa\u003c/em\u003e is the hatching rate of normal male moths paired with normal female moths. \u003cem\u003eHs\u003c/em\u003e is the hatching rate of sterile male moths paired with normal female moths. \u003cem\u003eEe\u003c/em\u003e is the hatching rate of mixed male moths of a certain S/N ratio paired with normal female moths. \u003cem\u003eE\u003c/em\u003e is the expected hatching rate of a mixed S/N ratio male moth paired with a normal female moth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Effect of linalool fumigation on mating competitiveness of \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThree disposable transparent plastic boxes (21*14*7 cm) with a volume of 1500 mL each were assembled for experimentation. A strip of filter paper (1 cm in wide and 5 cm long) was suspended in the center of the lid of each box. Male moths that newly emerged were introduced into the boxes. Subsequently, linalool was evenly distributed onto the filter paper strips within the boxes to achieve concentrations of 70, 83, and 96 \u0026micro;L/m\u0026sup3;, which were clearly labeled on the lids of the respective boxes. After a 3-hour fumigation period, the moths were removed and the males and females from the same day were segregated into adult mating boxes at a 1:1 ratio. The assessment procedures remained consistent with those described in section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Effect of linalool fumigation on mating competitiveness of sterile \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo investigate the effect of linalool fumigation on mating competitiveness of sterile \u003cem\u003eC. pomonella\u003c/em\u003e in laboratory, the 8-day-old male pupae were irradiated with X-rays at a dose of 200 Gy, subsequently maintained in a growth chamber until emergence. The freshly emerged male adults underwent a 3-hour fumigation with linalool. The procedures for fumigation, assessment of average survival time, fecundity per female, egg hatchability, mating frequency, and mating competitiveness followed the protocols described above-mentioned.\u003c/p\u003e \u003cp\u003eTo investigate the effect of linalool fumigation on mating competitiveness of sterile \u003cem\u003eC. pomonella\u003c/em\u003e in orchard, a field trial was conducted at Wumeifang Village, Jiuzhai Town, Gaizhou City, Yingkou City, Liaoning Province (40\u0026deg;3\u0026prime;52 \"N, 122\u0026deg;5\u0026prime;44 \"E) from August 1 to September 2, 2023. Small insect rearing nets (60 cm*60 cm*90 cm) with 40 mesh were affixed to tree trunks, and adult mating age were placed inside these nets for fumigation purposes. The experimental procedures for evaluating mean longevity, single female fecundity, hatching rate, mating rate, and mating competitiveness were consistent with those described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Effects of linalool fumigation on the flight ability of sterile \u003cem\u003eC. pomonella\u003c/em\u003e moths\u003c/h2\u003e \u003cp\u003eThe flight performance of moths was assessed using the FXMD-24-USB insect flight information system (Henan Hebi Jiaduoke Industry and Trade Co., Ltd, Hebi, Henan, China), according to the method described in Huang et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Normal, sterile, and fumigated sterile male moths were segregated into distinct adult rearing boxes and provided with a daily diet of 10% honey water. Then, 25 healthy male moths with intact wings that were 3 days post-emergence were selected for the study, and the flight capability of each group was evaluated. The insects were initially immobilized on a chilled surface before their abdomens were exposed. A copper wire was fashioned into a loop, folded at a 90\u0026deg; angle, and affixed to the moth's abdomen using a small amount of adhesive, ensuring proper fixation by inflation and rapid solidification of the glue. The other end of the wire was secured to the arm of the flight mill, positioning the moth parallel to the ground and aligning its flight direction with the mill. Subsequently, the insect flight information system was activated to input data on the test subjects. Flight ability assessments were conducted from 22:00 to 6:00 in a darkened environment, maintaining a constant ambient temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃ and relative humidity of 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5% throughout the evaluation process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 RNA isolation and cDNA synthesis\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from a collective of ten 3-day-old male adults of \u003cem\u003eC. pomonell\u003c/em\u003ea, who were subjected to either 200 Gy X-ray irradiation or not, utilizing the EAStep Super Total RNA Extraction Kit (Promega, Shanghai, China) in accordance with the manufacturer's guidelines. The concentration and quality of the RNA samples were assessed employing a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, 1 \u0026micro;g of RNA from each treatment was utilized in the synthesis of complementary DNA (cDNA) using the GoScript\u0026trade; Reverse Transcription Mix (Promega, Madison, WI, USA). The resulting cDNA was preserved at -20\u0026deg;C until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Reverse-transcription quantitative polymerase chain reaction analysis\u003c/h2\u003e \u003cp\u003eThe mRNA levels of pheromone recognition-related genes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S8) were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR) with Bio-Rad CFX96 (Bio-Rad, Hercules, CA, USA) in a reaction of 20 \u0026micro;L, containing of 1 \u0026micro;L of cDNA template, 10 \u0026micro;L of TB Green Premix Ex Taq 2 (TliRNaseH Plus) (TaKaRa, Dalian, China), 1 \u0026micro;L of each primer (10 \u0026micro;mol/L), and 7 \u0026micro;L double-distilled water (ddH2O). The RT-qPCR cycled at 95\u0026deg;C for 30 s, followed by 40 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 30 s. Afterward, a melting curve was conducted starting at 55\u0026ndash;95\u0026deg;C to identify the specificity of PCR products. The internal control genes used were \u003cem\u003eEF-1α\u003c/em\u003e (MN037793) and \u003cem\u003eβ-Actin\u003c/em\u003e (KC832921) of \u003cem\u003eC. pomonella\u003c/em\u003e (Wei et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For each three biological replicates and three technical replicates were conducted. Relative expression levels for each gene were calculated using the 2\u003csup\u003e\u0026minus;△△CT\u003c/sup\u003e method (Livak and Schmittgen, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Suppression of field population of \u003cem\u003eC. pomonella\u003c/em\u003e by pilot release of sterile males\u003c/h2\u003e \u003cp\u003ePilot release of sterile males were conducted between July 8 and September 2, 2023, in Wumeifang Village, Jiuzhai Town, Gaizhou City, Yingkou City, Liaoning Province, China (40\u0026deg;3\u0026prime;52 \"N, 122\u0026deg;5\u0026prime;44 \"E). Prior to release, a central tree within the orchard was designated and marked, with sex traps positioned at 5-meter intervals in the cardinal directions of east, south, west, and north from the central tree. Sterile males were gently coated with fluorescent powder, distinguishing fumigated and unfumigated specimens by color before being released at 18:00. The control orchard, devoid of sterile male releases, was equipped with 5 trap sets, with trapped moth counts recorded at 10-day intervals post-release. Additionally, daily temperature and precipitation data were collected throughout the release period.\u003c/p\u003e \u003cp\u003eThe five-point sampling method was employed in both the release orchard and control orchard to designate five points within the central and perimeter areas of the orchard. Around each of these points, five trees were meticulously selected and distinctly labeled. Prior to the release, a comprehensive examination of the total fruit count and the specific number of fruits was conducted. Subsequently, at intervals of 10 days, the quantity of infested fruits was meticulously documented, allowing for the calculation of the fruit infestation rate. This rate was determined by dividing the total number of infested fruits by the overall number of fruits examined, expressed as a percentage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Data analysis\u003c/h2\u003e \u003cp\u003eThe statistical significance of various biological parameters of \u003cem\u003eC. pomonella\u003c/em\u003e, encompassing metrics such as average emergence rate, longevity, fecundity, sterility rate, mating rate, as well as the flight characteristics of males including flight time, distance, and speed, along with gene expression in controlled laboratory settings and dispersal distance within the orchard, were meticulously analyzed utilizing the one-way analysis of variance (ANOVA) coupled with Duncan's test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) through SPSS 19 software (IBM Corporation, Armonk, NY, USA). Furthermore, significant difference in total number of moths trapped per trap, amount of insects trapped and fruit decay rate was analyzed through ANOVA with independent samples t-tests (*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) using SPSS 19. The results were graphically represented using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA), with all values being expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Additionally, the sterility rate of \u003cem\u003eC. pomonella\u003c/em\u003e under varying X-ray dosage levels was modeled using a logistic curve in Origin 2019 software (Origin Lab Corporation, Northampton, MA 02115, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of different doses of X-ray irradiation on the biological parameters of \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe effects of different doses of X-ray irradiation on the physiological traits of \u003cem\u003eC. pomonella\u003c/em\u003e were explored in a series of experiments that examined the emergence rate, lifespan, reproductive capacity, and sterility of 8-day-old pupae exposed to varying levels of X-ray radiation. The results indicated that there was no statistically significant variance in the emergence rate of male moths \u003cem\u003eC. pomonella\u003c/em\u003e within the range of 50\u0026ndash;250 Gy compared to the control group. Notably, a reduction in the emergence rate of male \u003cem\u003eC. pomonella\u003c/em\u003e moths was evident at 300 Gy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). However, the survival rate of moths arising from male pupae irradiated with various doses of radiation did not exhibit a significant difference compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Furthermore, a decrease in lifespan was evident with X-ray exposure surpassing 200 Gy when compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Fecundity levels remained steady across different radiation levels compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Additionally, an escalation in sterility rate was observed at doses below 250 Gy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), while a reduction in mating rate was noted at doses exceeding 200 Gy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e3.2 Effect of 200 Gy X-ray irradiation on mating competitiveness of male \u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e3. 2 Effect of 200 Gy X-ray irradiation on mating competitiveness of male \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2.1 Laboratory test\u003c/h2\u003e \u003cp\u003eThe hatching rate of eggs for SM:NM:NF at the 1:0:1 ratio was found to be 12.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51%, a value significantly lower compared to the ratios of 0:1:1 (72.96\u0026thinsp;\u0026plusmn;\u0026thinsp;7.31%) and 1:1:1 (64.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.95%). When male adults were combined with different ratios and matched with normal female adults, the expected hatching rate (E) was 42.77%. The competitive mating index (C) was determined to be 0.17, indicating that the mating competitiveness of male moths was reduced by 200 Gy of irradiation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e200Gy X-ray irradiation on the mating competitiveness of \u003cem\u003eC. pomonella\u003c/em\u003e adults\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExperimental site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMatching ratio\u003c/p\u003e \u003cp\u003e(S♂: N♂: N♀)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFecundity\u003c/p\u003e \u003cp\u003e(eggs laid per female)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eHatching rate/%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompetitive capacity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eObserved value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExpected value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIndoor experiment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e116.20\u0026thinsp;\u0026plusmn;\u0026thinsp;19.97 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.96\u0026thinsp;\u0026plusmn;\u0026thinsp;7.31 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 0 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103.75\u0026thinsp;\u0026plusmn;\u0026thinsp;4.42 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e106.07\u0026thinsp;\u0026plusmn;\u0026thinsp;5.80 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.95 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eField experiment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.45\u0026thinsp;\u0026plusmn;\u0026thinsp;4.09 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.16\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 0 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.13\u0026thinsp;\u0026plusmn;\u0026thinsp;10.60 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.46 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54.26\u0026thinsp;\u0026plusmn;\u0026thinsp;6.81 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.096\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eData are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. N: Number of unirradiated males; S: Number of irradiated males.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Orchard trial\u003c/h2\u003e \u003cp\u003eThe hatching rate of eggs for the SM:NM:NF group at the 1:0:1 ratio was observed to be 10.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.46%, a significantly lower rate compared to the 0:1:1 ratio (64.16\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11%) and the 1:1:1 ratio (59.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.47%). When male adults were introduced in various ratios and paired with normal female adults, the expected hatching value (E) was calculated to be 37.49%. Additionally, the competitive mating index (C) was determined to be 0.096, suggesting that a radiation dosage of 200 Gy diminished the mating competitiveness of male moths (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects of different concentrations of linalool fumigation on biological parameters of \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo elucidate the impact of varying concentrations of linalool fumigation treatments on the biological parameters of \u003cem\u003eC. pomonella\u003c/em\u003e, experimental investigations were conducted to assess the survival duration, oviposition, egg hatching efficacy, and mating activity of male moths. The results revealed no notable disparities in the aforementioned parameters among male moths subjected to distinct levels of linalool fumigation in comparison to the control cohort, indicating that the application of linalool fumigation did not adversely affect the male moths (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effects of different concentrations of linalool fumigation on mating competitiveness of \u003cem\u003eC. pomonella\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe hatching rate of eggs resulting from the pairing of fumigation-treated normal males with normal females were determined to be 73.63\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86 percent, while the pairing of untreated normal males with normal females yielded a hatching rate of 71.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74%. When comparing the hatching rate of fumigation-treated normal males to the overall hatching rate, the value was found to be 72.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33%. There was no significant difference observed in the hatchability of fecundity across three different mating combinations, with an average rate of 72.52%. Furthermore, the mating competitiveness index of \u003cem\u003eC. pomonella\u003c/em\u003e normal males that were exposed to linalool fumigation at a concentration of 70\u0026micro;L/m\u0026sup3;was calculated to be 1.06, suggesting an enhancement in the mating competitiveness of the males (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of different concentrations of linalool fumigation on the mating competitiveness of \u003cem\u003eC. pomonella\u003c/em\u003e males\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFumigation concentration\u003c/p\u003e \u003cp\u003e(\u0026micro;L/m\u0026sup3; )\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMatching ratio\u003c/p\u003e \u003cp\u003e(F♂: N♂: N♀)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFecundity\u003c/p\u003e \u003cp\u003e(eggs laid per female)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eHatching rate/%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompetitive capacity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eObserved value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExpected value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 0 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.46\u0026thinsp;\u0026plusmn;\u0026thinsp;9.51 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73.63\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.74\u0026thinsp;\u0026plusmn;\u0026thinsp;9.33 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e72.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 0 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94.75\u0026thinsp;\u0026plusmn;\u0026thinsp;13.79 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e78.47\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e74.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 0 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.98 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.83 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe percentage of eggs hatched when 83 \u0026micro;L/m\u0026sup3; of linalool was used to fumigate normal males paired with normal females was recorded at 78.47\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28%. For normal males paired with normal females, the hatching rate was slightly lower at 71.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74%. When fumigated normal males were paired with normal females, the hatching rate was 75.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43%. There was no significant difference in the hatching rate of eggs produced by the three different mating combinations, all averaging around the expected value of 74.94. The mating competitiveness index of males treated with linalool at 83 \u0026micro;L/m\u0026sup3;was 1.25, indicating an improvement in mating competitiveness compared to those treated at 70 \u0026micro;L/m\u0026sup3; (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe egg hatching rate of 96 \u0026micro;L/m\u0026sup3; linalool-exposed normal male moths when paired with normal females was determined to be 76.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36%, while that of normal males paired with normal females was 71.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74%, and the hatching rate of fumigated normal males when paired with other males and females was 74.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.83%. These results indicate that there were no significant differences in hatching rates among the three mating combinations studied, all of which were close to the anticipated value of 73.97%. Furthermore, the mating competitiveness index of males exposed to linalool at 96\u0026micro;L/m\u0026sup3;was calculated to be 1.24, suggesting a reduced mating competitiveness compared to males exposed to linalool at 83 \u0026micro;L/m\u0026sup3; (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effect of linalool fumigation on mating competitiveness of sterilized male \u003cem\u003eC. pomonella\u003c/em\u003e moths\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 Laboratory test\u003c/h2\u003e \u003cp\u003eThe results revealed that male moths exposed to 200 Gy X-rays exhibited notably reduced hatching and mating frequencies in comparison to the untreated group. Notably, there were no substantial variations in any of the outcomes between the fumigated and non-fumigated sterilized males, indicating that the linalool fumigation did not impact the reproductive characteristics of sterilized males (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSpecifically, the hatching rate of linalool-fumigated irradiated sterilized males that were paired with normal females was 13.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33%, while that of irradiated sterilized males paired with normal females stood at 10.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41%. Moreover, the hatching rate of fumigation-treated irradiated sterilized males when paired with females was 12.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98%, suggesting no significant variance in the hatching rate of eggs produced by the three different pairings (F\u0026thinsp;=\u0026thinsp;0.305, df\u0026thinsp;=\u0026thinsp;8, P\u0026thinsp;=\u0026thinsp;0.748), with an expected value of 12.21%. The mating competition index of 1.11 for the irradiated sterilized males of moths fumigated with linalool indicated an enhancement in the mating competitiveness of the irradiated sterilized males due to linalool fumigation (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of different concentrations of linalool fumigation on the mating competitiveness of 200 Gy X-ray sterilized \u003cem\u003eC. pomonella\u003c/em\u003e males\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExperimental site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMatching ratio\u003c/p\u003e \u003cp\u003e(F♂: N♂: N♀)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFecundity(eggs laid per female)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eHatching rate/%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompetitive capacity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eObserved value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExpected value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIndoor experiment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e106.54\u0026thinsp;\u0026plusmn;\u0026thinsp;5.71 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 0 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103.30\u0026thinsp;\u0026plusmn;\u0026thinsp;12.00 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94.35\u0026thinsp;\u0026plusmn;\u0026thinsp;11.43 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eField experiment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60.13\u0026thinsp;\u0026plusmn;\u0026thinsp;10.60 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.46 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 0 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.40\u0026thinsp;\u0026plusmn;\u0026thinsp;5.28 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 : 10 : 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.66\u0026thinsp;\u0026plusmn;\u0026thinsp;4.09 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2 Orchard trial\u003c/h2\u003e \u003cp\u003eThe results of the field experiment revealed a significant decrease in the longevity and fecundity of sterile males of \u003cem\u003eC. pomonella\u003c/em\u003e compared to the control group. Interestingly, there was no notable variance between the biological parameters of linalool-fumigated sterile males and non-fumigated ones, suggesting that the fumigation treatments did not impact these parameters within the field environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen linalool-fumigated irradiated sterile males were paired with normal females, the hatchability was observed to be 14.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89%, while it was 10.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.46% for \u003cem\u003eC. pomonella\u003c/em\u003e irradiated sterile males mated with normal females. Moreover, the hatchability of fumigation-treated irradiated sterile males when paired with females stood at 12.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98%. These findings indicate that there was no significant difference in hatchability and fecundity among the three different combinations (F\u0026thinsp;=\u0026thinsp;0.249, df\u0026thinsp;=\u0026thinsp;8, P\u0026thinsp;=\u0026thinsp;0.756), with an average value of 12.90%. The mating competition index of 1.06 for the linalool-fumigated irradiated sterile males of \u003cem\u003eC. pomonella\u003c/em\u003e suggests that linalool fumigation enhanced the mating competitiveness of the irradiated sterile males (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effects of linalool fumigation on the flight ability of sterile \u003cem\u003eC. pomonella\u003c/em\u003e moths\u003c/h2\u003e \u003cp\u003eThe flight duration of 3-day-old male adults, sterile males, and fumigation treated males was 53.82\u0026thinsp;\u0026plusmn;\u0026thinsp;7.17, 5.61\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36, and 10.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46 min, respectively. While no significant disparity was observed in the flight duration between sterile males and fumigation treated males, both groups exhibited significantly shorter flight times compared to the control group (F\u0026thinsp;=\u0026thinsp;30.820, df\u0026thinsp;=\u0026thinsp;44, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Moreover, there were no significant differences in flight distance or flight speed among the treatment groups when compared to the control group, (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.7 Effects of linalool fumigation on the expression levels of sex pheromone receptor-related genes in sterile\u003c/b\u003e \u003cb\u003eC. pomonella\u003c/b\u003e \u003cb\u003emoths\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRT-qPCR results showed that \u003cem\u003eCpomOR1, CpomOR2a, CpomOR3a, CpomOR3b, CpomOR5, CpomOR6a, CpomOR7, CpomGOBP1\u003c/em\u003e, and \u003cem\u003eCpomGOBP2\u003c/em\u003e genes were significantly reduced in \u003cem\u003eC. pomonella\u003c/em\u003e male moths that emerged from pupae irradiated with 200 Gy X-ray (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression levels of \u003cem\u003eCpomOR3a, CpomOR3b\u003c/em\u003e, and \u003cem\u003eCpomOR5\u003c/em\u003e were significantly rebounded after linalool fumigation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Suppression of field population of \u003cem\u003eC. pomonella\u003c/em\u003e by pilot release of sterile males\u003c/h2\u003e \u003cp\u003eA total of 5342 sterile males were released, with 2735 of them being released from linalool fumigated moths. Among these, 60.44\u0026thinsp;\u0026plusmn;\u0026thinsp;4.48% of males flew out normally, while 6.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51% fell to the ground post take-off, leaving 39.56\u0026thinsp;\u0026plusmn;\u0026thinsp;4.48% unable to take off normally (Table S2). The percentage of males falling to the ground was 59.84\u0026thinsp;\u0026plusmn;\u0026thinsp;6.86%. Additionally, 2607 sterile males of \u003cem\u003eC. pomonella\u003c/em\u003e without linalool fumigation were also released, where 62.17\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25% flew out normally, 17.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86% fell to the ground after take-off, and 37.83\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25% couldn't take off normally. Notably, 78.19\u0026thinsp;\u0026plusmn;\u0026thinsp;6.36% of males picked with tweezers failed to take off and fell to the ground (Table S3).\u003c/p\u003e \u003cp\u003eThroughout the experiment, the ambient temperature at the experimental site exhibited minimal variations, with a singular precipitation event exceeding 100 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Results revealed that, at a 5-meter distance, the recapture of sterile male moths in traps averaged 12.25\u0026thinsp;\u0026plusmn;\u0026thinsp;3.07, whereas the recapture of fumigation-treated sterile males was 8.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16. Similarly, at distances of 10 m, the recaptures were 10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78 and 9.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18, respectively. At 15 m, the retrievals were 8.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55 and 6.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19, respectively. Finally, at a distance of 20 m, the retrievals were 0\u0026thinsp;\u0026plusmn;\u0026thinsp;0 and 1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65, respectively. The results indicated that there were no statistically significant variations in the recaptures of sterile males and fumigation-treated sterile males of \u003cem\u003eC. pomonella\u003c/em\u003e at the 10 and 20 m distances at a significance level of 0.05 (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, notable differences were noted in recaptures at 5 and 15 meters, with statistically significant variances observed at the 0.05 level (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe deployment of \u003cem\u003eC. pomonella\u003c/em\u003e sterile males over a period of two months resulted in a notable decline in fruit infestation within the treated orchards, registering at 24.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17%. This rate was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in contrast to the control orchard where infestation levels reached 34.01\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87%. These findings provide evidence that the introduction of sterile \u003cem\u003eC. pomonella\u003c/em\u003e males effectively mitigated fruit infestation occurrences, thereby diminishing both the incidence of infested fruits and the overall infestation rate within the orchard (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe captures of wild male moths did not exhibit a notable variance between the release area and the control from July 7 to July 14, 2023. Nonetheless, subsequent to July 31, 2023, a noteworthy decline in captures within the release area was observed, presenting a significant contrast with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Based on these findings, it is evident that the introduction of sterile male \u003cem\u003eC. pomonella\u003c/em\u003e moths in the natural environment substantially reduces the population of wild male moths (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe radiation dose is a critical factor in achieving the desired sterility level of insects in the SIT program (Proverbs and Newton, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). In this study, we demonstrated that the biological traits of male \u003cem\u003eC. pomonella\u003c/em\u003e varied in their decline with increasing levels of irradiation. This observation is consistent with a previous study on \u003cem\u003eGlossina palpalis\u003c/em\u003e tsetse flies, which showed that higher radiation doses reduced the fecundity of females (Pagabeleguem et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, we observed a significant decrease in the mating rate of sterile male \u003cem\u003eC. pomonella\u003c/em\u003e with normal females when exposed to radiation above 200 Gy, mirroring the findings in \u003cem\u003eG. palpalis\u003c/em\u003e (Pagabeleguem et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The OK SIR program, designed to control \u003cem\u003eC. pomonella\u003c/em\u003e populations, initially utilized 250 Gy of \u003csup\u003e60\u003c/sup\u003eCo-γ irradiation, resulting in male sterility rates of 85\u0026ndash;92% (Holm, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Since 2014, a consistent dose of 200 Gy has been employed to balance sterility levels and mating competitiveness (Thistlewood and Judd, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To optimize the trade-off between male sterility and minimize the negative effects of radiation on the insects, it is recommended to use 200 Gy as the optimal dose for inducing sterility in males \u003cem\u003eC. pomonella\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe mating competitiveness of sterile insects is crucial for their ability to effectively compete with wild males for mating opportunities with females (Vreysen et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Vreysen and Robinson, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Previous research has shown that inadequate dosages may result in incomplete sterilization, while excessive irradiation can reduce the mating competitiveness of released insects compared to wild counterparts, thus affecting the success of SIT programs (Parker et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, maintaining a delicate balance between mating competitiveness and sterility is essential during copulatory interactions with wild female insects (Bakri et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Evaluating the optimal irradiation dose is critical to assessing the mating competitiveness of male insects bred for this purpose (Parker et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, reduced mating competitiveness is commonly observed in irradiated \u003cem\u003eC. pomonella\u003c/em\u003e populations (North, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1975\u003c/span\u003e). Jiang et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) also reported a significant decrease in the mating competitiveness of male \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e following exposure to 250 Gy of X-ray irradiation. In our recent studies, male \u003cem\u003eC. pomonella\u003c/em\u003e pupae irradiated with 366 Gy of X-rays exhibited mating competitiveness indices of 0.001 in laboratory (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea) and 0.0088 in orchards (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003eb). In this study, when subjected to 200 Gy of X-ray irradiation, the mating competitiveness index of sterilized male \u003cem\u003eC. pomonella\u003c/em\u003e decreased, indicating a decline in mating competitiveness post-irradiation. Nevertheless, compared to the impact of 366 Gy irradiation, the mating competitiveness index of \u003cem\u003eC. pomonella\u003c/em\u003e males following 200 Gy irradiation increased to 0.17 in laboratory conditions and 0.096 in orchards, suggesting that an optimized irradiation dose can enhance the mating competitiveness of sterile insects to a certain degree. However, the mating competitiveness of \u003cem\u003eC. pomonella\u003c/em\u003e males post-exposure to 200 Gy irradiation persists at a sub-optimal level. Therefore, it is imperative to prioritize efforts aimed at improving the mating competitiveness of sterile males in future studies.\u003c/p\u003e \u003cp\u003eThe diminished mating competitiveness evident in irradiated insects may stem from the compromised olfactory system in male insects, leading to a reduced ability to perceive female sex pheromones. To investigate this hypothesis, linalool, a volatile compound found in host plants known to enhance the attraction of \u003cem\u003eC. capitata\u003c/em\u003e moths (Casado et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), was utilized to fumigate male \u003cem\u003eC. pomonella\u003c/em\u003e moths. The findings indicated that although linalool fumigation did not affect longevity, fecundity, hatching rates, or overall mating performance, it did enhance the mating competitiveness of male moths. Another investigation on \u003cem\u003eC. capitata\u003c/em\u003e demonstrated that the mating competitiveness of normal male adults and mass-reared irradiated males increased subsequent to fumigation with ginger root oil (GRO), compared to non-fumigated adults. Notably, GRO fumigation did not have any notable impacts on the body weight or longevity of irradiated mass-reared males (Shelly and McInnis, 2001). These results suggest that linalool fumigation might potentially mitigate male sterility induced by X-ray irradiation, possibly due to enhanced male sex pheromone communication in the antennae of male \u003cem\u003eC. pomonella\u003c/em\u003e moths, as observed in \u003cem\u003eC. capitata\u003c/em\u003e post ginger root oil fumigation (Papadopoulos et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe olfactory system plays a vital role in various insect behaviors like feeding, orientation, host-seeking, mating, and oviposition (Li et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Chemoreception in insects is a complex process involving various chemosensory genes, such as odorant receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs), odorant-binding proteins (OBPs), chemosensory proteins (CSPs), and sensory neuron membrane proteins (SNMPs) (Yang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In particular, the importance of chemosensory genes in recognizing pheromones in \u003cem\u003eC. pomonella\u003c/em\u003e has been extensively researched. Tian et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have identified male pheromone receptor genes in \u003cem\u003eC. pomonella\u003c/em\u003e, including \u003cem\u003eCpomOR1\u003c/em\u003e, \u003cem\u003eCpomOR2a\u003c/em\u003e, \u003cem\u003eCpomOR3\u003c/em\u003e, \u003cem\u003eCpomOR5\u003c/em\u003e, \u003cem\u003eCpomOR6a\u003c/em\u003e, and \u003cem\u003eCpomOR7\u003c/em\u003e, through transcriptome analysis of the antenna. Knock-out the \u003cem\u003eCpomOR1\u003c/em\u003e gene using CRISPR/Cas9 technology has been shown to impact the fertility and fecundity of \u003cem\u003eC. pomonella\u003c/em\u003e (Garczynski et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, \u003cem\u003eCpomOR3a\u003c/em\u003e and \u003cem\u003eCpomOR3b\u003c/em\u003e genes in \u003cem\u003eC. pomonella\u003c/em\u003e are involved in recognizing plant volatiles and the sex pheromone codlemone, thereby enhancing the moths' response to codlemone (Wan et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, we revealed a decrease in gene expression in \u003cem\u003eC. pomonella\u003c/em\u003e males' antenna following 200 Gy of X-ray irradiation, suggesting a reduced sex pheromones response in male moths. Conversely, an increase in the expression of \u003cem\u003eCpomOR3a\u003c/em\u003e, \u003cem\u003eCpomOR3b\u003c/em\u003e, and \u003cem\u003eCpomOR5\u003c/em\u003e genes was observed after linalool fumigation, enhancing the recognition of sex pheromones.These findings support the efficacy of linalool fumigation in mitigating male sterility induced by X-ray irradiation, likely through the upregulation of essential chemosensory genes and the activation of pheromone recognition mechanisms in \u003cem\u003eC. pomonella\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that the release of \u003cem\u003eC. pomonella\u003c/em\u003e males into the pear orchard following exposure to 366 Gy X-ray did not affect the wild populations or the fruit decay rate (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, we carried out a small-scale trial in different fields utilizing the SI) to manage \u003cem\u003eC. pomonella\u003c/em\u003e populations. Our study demonstrated that by releasing three successive batches of sterile male \u003cem\u003eC. pomonella\u003c/em\u003e moths irradiated at 200 Gy, along with linalool fumigation, in the specified orchard area, there was a substantial decrease in \u003cem\u003eC. pomonella\u003c/em\u003e infestations in the apple crop. This reduction was evident through significantly lower infestation rates compared to the control orchard. Additionally, field observations indicated a notable decrease in the recapture rate of wild \u003cem\u003eC. pomonella\u003c/em\u003e moths in comparison to the control group. These results suggest that the utilization of sterile males effectively suppressed the wild \u003cem\u003eC. pomonella\u003c/em\u003e population in the orchards, consequently reducing fruit damage. The findings highlight an enhancement in the field application of the SIT for \u003cem\u003eC. pomonella\u003c/em\u003e control after optimizing the irradiation dose.\u003c/p\u003e \u003cp\u003eIn summary, we have identified the optimal sterilizing dose of X-ray irradiation for \u003cem\u003eC. pomonella\u003c/em\u003e and introduced a plant volatiles fumigation approach to enhance the mating competitiveness of sterilized males. Additionally, we investigated the effects of irradiation and fumigation on the expression of genes related to pheromone recognition in the male antennal. Notably, our study demonstrated the successful reduction of the \u003cem\u003eC. pomonella\u003c/em\u003e population in the orchards through repeated releases of sterilized males in conjunction with linalool fumigation on a pilot scale. These findings offer valuable insights for the implementation of the SIT as a strategy of controlling \u003cem\u003eC. pomonella\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSheng-Wang Huang: software, methodology, investigation, formal analysis, writing original draft, Peng-Cheng Wang: software, methodology, investigation, formal analysisYan Wang: software, review and editing. Jie-Qiong: formal analysis, conceptualization. Ping Gao: writing, review, and editing, resources.Xue-Qing Yang: supervision, resources, project administration, funding acquisition, conceptualization, writing, review, and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was funded by the National Key R\u0026amp;D Program of China (2021YFD1400200, 2023YFD1400704) and the IAEA TC program (CPR5027).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnisimov AI, Lazurkina NV, and Shvedov AN (1989) Influence of radiation-induced genetic damage on the suppressive effect of inherited sterility in the codling moth (Lepidoptera: Tortricidae). Ann. Entomol. Soc. 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Food Chem. 68 (21): 5825\u0026ndash;5834. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphys.2022.989006\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2022.989006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eProverbs MD, Newton JR (1962) Influence of gamma radiation on the development and fertility of the codling moth, Carpocapsa pomonella (L.) (Lepidoptera: Olethreutidae). Can. J. Zool, 40: 401\u0026ndash;419. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1139/z62-038\u003c/span\u003e\u003cspan address=\"10.1139/z62-038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNorth DT, (1975) Inherited sterility in lepidoptera. Annu. Rev. Entomol. 20:167\u0026ndash;182. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1051/agro/2010009\u003c/span\u003e\u003cspan address=\"10.1051/agro/2010009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, J. et al. (2019) The olfactory coreceptor IR8a governs larval feces-mediated competition avoidance in a hawkmoth. Proc. Natl. Acad. Sci. USA 116: 21828\u0026ndash;21833. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1913485116\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1913485116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Mo BT, Li GC et al (2024) Identification and functional characterization of chemosensory genes in olfactory and taste organs of \u003cem\u003eSpodoptera litura\u003c/em\u003e (Lepidoptera: Noctuidae). Insect Sci. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1744-7917.13350\u003c/span\u003e\u003cspan address=\"10.1111/1744-7917.13350\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Huang S, Zhao S, et al (2023) The effect of X-ray irradiation on the fitness and field adaptability of the codling moth: an orchard study in Northeast China. Insects 14(7): 615. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects14070615\u003c/span\u003e\u003cspan address=\"10.3390/insects14070615\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sterile insect technique, Codling moth, X-ray, Iirradiation, Mating competitiveness, Plant volatile","lastPublishedDoi":"10.21203/rs.3.rs-4677671/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4677671/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe implementation of sterile insect technique (SIT) has proven effective in the area-wide suppression of several significant agricultural and sanitary pests by employing traditional cobalt-60 (\u003csup\u003e60\u003c/sup\u003e Co-γ) as a radiation source. Recently, X-ray has been validated as a feasible alternative to \u003csup\u003e60\u003c/sup\u003e Co-γ radiation sources. Nonetheless, higher doses of X-ray irradiation lead to insect sterility but diminish mating competitiveness, thereby impacting the effectiveness of SIT applications. Thus, it is crucial to ascertain the optimal irradiation dose and develop strategies to enhance the mating competitiveness of sterile insects to enhance SIT efficacy. In this study, we determined the effect of various X-ray irradiation doses (ranging from 0 to 366 Gy) on the fecundity, fertility, and mating competitiveness of \u003cem\u003eCydia pomonella\u003c/em\u003e, a globally invasive fruit pest. Results demonstrated that the sterility rate of sterile males increased proportionally with irradiation dose up to 200 Gy, beyond which it plateaued. Notably, exposure to 200 Gy of irradiation notably decreased the mating competitiveness of male, as evidenced by a mating competitiveness index of 0.17 in laboratory and 0.096 in the orchard. This decline in mating competitiveness is likely linked to the down-regulation of genes associated with the recognition of sex pheromones, specifically \u003cem\u003eCpomOR3a\u003c/em\u003e, \u003cem\u003eCpomOR3b\u003c/em\u003e, and \u003cem\u003eCpomOR5\u003c/em\u003e, following X-ray irradiation. Fumigation of the plant volatile, linalool at varying concentrations (70, 83, and 96 \u0026micro;L/m\u0026sup3;) resulted in differential enhancements in male mating competitiveness, with the moderate concentration significantly improving the competitiveness of sterilized males, possibly by restoring their ability to recognize sex pheromones. Implementation of repeated releases of sterilized males on a pilot scale led to a notable reduction in the population of \u003cem\u003eC. pomonella\u003c/em\u003e in the field. These findings indicate that fumigation with plant volatiles has the potential to mitigate male sterility induced by X-ray irradiation, offering a promising approach to enhance the efficacy of SIT applications for the control of \u003cem\u003eC. pomonella\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Plant volatile-based fumigation improves mating competitiveness of males for population suppression of the global fruit pest Cydia pomonella","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 18:08:32","doi":"10.21203/rs.3.rs-4677671/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"10d72bc5-6d01-46be-a3fa-cd08292c54eb","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-29T18:08:35+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 18:08:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4677671","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4677671","identity":"rs-4677671","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}