Females codling moths evade the mating disruption control tactic

Females codling moths evade the mating disruption control tactic | 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 Females codling moths evade the mating disruption control tactic Rakefet Sharon, Maor Tomer, Almog Avraham, Ally R Harari This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4396430/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 Mating disruption, an environmentally friendly pest-control method, utilizes synthetic sex pheromones to interfere with males' ability to locate females for mating. An increase in successful mate encounters can occur due to uneven distribution of synthetic pheromones in the field, allowing males to locate and mate with calling females in areas of low pheromone concentration and areas with larger pest populations, where random encounters between males and females are more likely to happen by chance. The study results demonstrate greater fruit damage and higher abundances of male and female codling moths ( Cydia pomonella) in the margins compared to the centers of mating disruption-treated apple orchards. We suggest larger pest populations build up at the orchard margins over the season due to the following behavioral cascade: Females flee areas of high pheromone concentration to reduce the perceived “competition” for mates, while mated females move to the margins to minimize future “competition” for offspring's resources. Males then track the higher female population at the margins by following the females' full pheromone blend. To our knowledge, this is the first study to offer an interpretation supported by empirical data for the puzzling phenomenon of higher fruit damage in field margins under mating disruption. Sex pheromone Female auto-detection Intra-specific competition Behavior Edge effect Figures Figure 1 Figure 2 Figure 3 Introduction Mating disruption is an environmentally safe pest-control method that employs synthetic sex pheromones to hamper males' ability to locate females. This method is used against various insect pests worldwide, with moths being the predominant target species. 1–7 The large-scale implementation of pheromone-based mating disruption for pest control offers an opportunity to observe evolution in action. 8 The success of the mating-disruption technique is contingent on both its immediate impact on the targeted pests in the treated area and its sustained, long-term reduction of the pest populations in the region. 4 The method is based on preventing encounters of male and female pests. However, both males and females share a common strong interest in reproducing. Consequently, the mating-disruption approach can affect the selection pressures imposed on the sexual communication system, as males and females persist in their efforts to breed. Indeed, although the method has been generally successfully applied for many years and multiple crops, recent observations point to the technique’s diminishing efficacy (see Harari and Sharon 9 for review). A local increase in mate encounters may arise from uneven distribution of the synthetic pheromones in a specific field, creating areas with an insufficient dose or no pheromones, where males can readily locate a calling female. 1,10 Uneven distribution of pheromone molecules typically occurs at the margins of pheromone-treated areas. 10 The pheromone concentration in these zones is diminished due to the cascade of its concentration to adjacent areas lacking the mating-disruption pheromones. Another plausible explanation for the decline of mating-disruption efficacy is the emergence of resistance to the method. 9 For example, after many years of consistent applications of mating disruption in cotton fields to combat the pink bollworm ( Pectinophora gossypiella ), accumulating evidence suggests that resistance to the method has evolved. In fields treated against this pest, a synthetic pheromone consisting of a blend of the pheromone’s two components in a 1:1 ratio had been deployed continually since early 1985. 11 Notably, a substantial number of pink bollworm females in treated fields altered the pheromone ratio, enabling conspecific males to locate their pheromones within the treated areas. 12 Pink bollworm males exposed to the mating-disruption pheromone were able to find these females by recognizing the change in their emitted pheromone. However, they encountered significantly fewer laboratory-reared calling females. 9 Similarly, following 14 years of continuous applications of mating disruption against the small tea leafroller, Adoxophyes honmai, in Japan, examination of the pheromone gland in females revealed a threefold increase in the quantity of one of the two pheromone components in females collected from treated as compared to control fields. 13 Importantly, this specific component is for mating disruption. Females may have augmented their pheromone output in an effort to compete with the elevated concentration of this component in the field, aiming to attract males. Nevertheless, the resistance observed in the small tea leafroller was linked to a decline in male sensitivity to this specific pheromone component. 13–15 A decline in the effectiveness of mating disruption may also be explained by behavioral adaptations aimed at evading pheromone-flooded areas and seeking a pheromone-reduced environment. In such places, females emitting pheromones can successfully attract males. The pheromone-evading behavior implies that females have the ability to detect their species-specific pheromone in their environment. 16,17 Female autodetection was first verified in 1977, when cabbage looper ( Trichoplusia ni ) females were caught in female-baited traps. 18 It is evident in their altered behavior in mating disruption-treated fields, as has been documented in various insects, primarily moths. Some moth species exhibit a delay in the onset of calling in the presence of conspecific sex pheromones, 19,20 others demonstrate an earlier calling for males, 21,22 while yet others extend the calling window in an attempt to attract late-coming males. 23 Autodetection of female pheromones also triggers dispersal in certain moth species. 21,24,25 However, in other cases, an excess of female pheromones has also been observed to attract females and increase infestations. 26 The suggestion is that movement away from a concentrated pheromone source, indicative of a high population of competing females, may enhance the likelihood of female mating. 10 This movement could also drive away mated females, thereby reducing resource competition among progeny. 27 These observations highlight the need for further investigation into the effects of flooding treated fields with species-specific sex pheromones on the dispersal of females, the males' reaction to dispersing females, and the resulting damage to yield. The commercialization of mating disruption against the codling moth, Cydia pomonella (Tortricidae), was initiated in the United States in 1991. 28 The method is still employed extensively worldwide. 29 In Israel, mating disruption has been used against the codling moth since 1995 and continues to be widely implemented in most apple orchards. 30 This paper tests the hypothesized behavioral changes in response to mating disruption: (i) the virgin females avoid “competition” by moving to the margin; (ii) the mated females avoid “competition” on resources by moving to the margin; (iii) males follow the higher population of females at the margin and have better mating success. Methods Sites The study was conducted in five commercial apple orchards in northern Israel in 2020–2021, where the mating-disruption method has been implemented for over 20 years: three plots in the Golan Heights (Merom Golan20, Merom Golan21, and Keshet) and two in the Upper Galilee (Yron and Sasa. The dominant cultivars were Granny Smith and Pink Lady, which were harvested in the autumn (September–October). We selected plots at the orchards' margins with at least one side that did not share a border with another apple orchard. Mating disruption against the codling moth was applied (CheckMate CM by vapor dispenser; Suterra LLC, Bend, OR, USA) in all orchards at a density of 500 dispensers ha -1 . For each plot, we defined the margins (2–3 rows from the edge, ~5–10 m) and the center (15–25 rows from the edge, ~100–150 m). In Keshet, the rows were not parallel to the margin, and the margin was therefore defined as the 2nd to 3rd tree (~5 m), and the center was defined as the 25th tree (100 m). Treatments and measured variables Infected fruit At apple harvest in 2021 and 2022, we collected 30 random fruits from each of the four trees in each of the orchards' margins and a similar number of fruits from each of the four trees in the center. Fruits were assigned as infected when they had traces of codling moth eggs or exit holes of larvae before pupation. The percentage of infected fruits was calculated for each tree in each orchard's margin and center. Two-way ANOVA was used to compare the means of infected fruit between the margin and center of the plots, followed by paired t-test for each plot. Monitoring codling moth populations During 2021, nine combo traps that attract male and female codling moths (PHEROCON CMDA COMBO + AA, TRÉCÉ, West Adair, OK, USA) were placed in the margin, and nine traps at the center of each plot. The traps, set in Merom Golan21 in March 2021 and Keshet in May 2021, were checked weekly until the end of May 2022. Collected moths were separated into males and females by their mating organs. Females were dissected to determine their mating status by the presence or absence of at least one full spermatophore in their bursa copulatrix. The percentage of mated females out of all collected female moths was calculated. The number of trapped males and females was compared between each plot's margin and center by paired t-test. Results Margin effect on infested fruit An examination of infected fruit during harvest in the apple orchards revealed significantly higher percentages of damaged fruit at the margins than at the center of the plots (two-way ANOVA: F critical = -4.2, DF = 1, P < 0.0001). The plots differed in damage level (ANOVA: F critical = -2.7, DF = 4, P < 0.001), with a consistently higher percentage of infected fruit at the margins compared to the center in all orchards (Fig. 1). Margin effect on codling moth populations Comparing the number of males and females captured in the combo traps (Fig. 2A) during the season of 2021 and the spring of 2022 at the margin and center of the plots of two orchards (Merom Golan21 and Keshet) revealed a higher number of trapped males at the margins vs. center of the plots (t-statistic = -1.7, DF = 47, P < 0.0001; and t-statistic = -3.8; DF = 38; P < 0.001, respectively). The number of trapped females was also higher in the margins than at the center of the two plots (t-statistic = -3.2, DF = 47, P < 0.005; and t-statistic = -2.8, DF = 38, P < 0.005, respectively), as were the percentages of mated females in the two plots (Fig. 2B) (t-statistic = -4.4, DF = 35; P < 0.0001; and t-statistic = -2.5, DF = 28, P < 0.01, respectively). Both males and females were present at the orchard margins throughout the entire season (April–September) (Fig. 3). In the center, however, only first-generation males and females were trapped; none were trapped later in the season (Fig. 3A–D). Mated females followed this pattern (Fig. 3H–I). Discussion Mating disruption, a prevalent agricultural practice, aims to reduce pest populations by manipulating their chemical communication and mating behaviors. Despite its widespread adoption, recent reports from farmers suggest a decline in its effectiveness. This study shows that the diminished efficacy of the method occurs primarily at the orchards' periphery, where the bulk of pest populations and associated damage are concentrated. The findings reveal a notable accumulation of codling moth populations, encompassing both virgin and mated females, males, and offspring, along the apple orchards' margins compared to the orchards' center. An excess of species-specific pheromones poses mating constraints that challenge both male and female moths to evolve or develop ecological adaptations that will facilitate reproduction. One such adaptive strategy involves altering the pheromone's characteristics, as evinced in studies with pink bollworm females 12 and the small tea leafroller females. 13 An alternative physiological mechanism to circumvent the effects of excessive pheromones involves adjusting the male’s response threshold—diminishing the response to the component that serves for mating disruption. 13 Minor behavioral adaptations may also enable male moths to evade the disruptive impact of excess pheromones on mate location. For instance, the male codling moth flies upwind, preferentially in the upper reaches of the tree canopy. This strategy allows it to detect females emitting pheromones at the canopy top, which may be above the area in which pheromone dispensers have been distributed. 4 Research has predominantly focused on the impact of mating-disruption methods on males' ability to locate females, 1,31 and to a lesser extent, the indirect effects on females, such as delayed mating. 32 However, a pheromone-saturated environment may also directly affect the female's behavior. 33 Documented evidence demonstrates that Lepidoptera and Coleoptera females can autodetect their pheromones. 16 Detecting their species-specific pheromones is advantageous when females compete for breeding opportunities, including mates and resources crucial for their offspring's survival and fitness. 34 The concentration of species-specific sex pheromones in their immediate surroundings may provide females with information on population size and the intensity of the expected competition. 35 Hence, the flooded pheromone environment resulting from mating disruption may provide a false cue of high population density, 36 implying intense competition for males and potential competition among offspring for resources. This expected competition may change the females’ dispersal pattern. Indeed, research has revealed a repellent response in both virgin and mated females of the European grape berry moth, Lobesia botrana , when exposed to their own or synthetic versions of their pheromones. 37 This is supported by the behavior of female grape root borers ( Vitacea polistiformis ) which exhibited a distinct bimodality in vertical distribution when exposed to pheromone treatments, positioning themselves outside the pheromone plume, either higher or lower than control females. By moving, the females exploited the greater air movement and presumably lower pheromone concentration in these areas (Pearson et al., 2004). It has been suggested that moving away from pheromone sources increases the likelihood of female mating success. 10,16 Mating disruption may trigger virgin females to move away from areas that are saturated with pheromones, where their ability to be located by mate-seeking males is hindered. Similarly, it may prompt mated females to disperse from regions with high population density, thereby mitigating resource competition among offspring. 10,16 In this study, we demonstrate that in apple orchards continuously treated with mating-disruption methods, a significantly higher number of virgin and mated females are found at the orchard margins compared to the center of the orchard. Based on the observed concurrent population reduction in the orchard's center and its increase at the margins, we suggest that virgin and mated females move to the margins following the cascade in the pheromone concentration, as suggested by Pearson et al.'s model. 10 We also suggest that the higher number of males caught in baited traps in the margins compared to the center results from the males following the calling females to zones with lower pheromone concentrations. Previous studies of various pest species and crops treated with mating disruption have demonstrated significantly higher damage in the margins. An alternative explanation for this increased damage has been attributed to the invasion of gravid females from nearby untreated hosts. 3,4,10,38,39 However, the possibility of gravid females migrating from the fields' outskirts to oviposit in the treated field has scarcely been tested. With the hypothesis of invasion of gravid females in mind, our study was conducted in plots devoid of potential host plants for the codling moth. Furthermore, the presence of a higher number of males at the orchard margins alongside virgin and gravid females does not support the suggested invasion of gravid females as the sole explanation. Rather, our results align more closely with the concept of repellent characteristics associated with high concentrations of species-specific pheromones, as proposed by Pearson et al.'s model 10 and demonstrated by Koutsoumpeli et al. 37 and others. 21,24,25 Accordingly, virgin females move to the margins to reduce the perceived “competition” for mates, while mated females move to the margins to minimize future “competition” for their offspring's resources. Males likely join the higher female population at the margin by following the high concentration of their full-blend pheromone. This is supported by the presence of first-generation male and female moths (April–May) in both the center and margins of the plots, with the population increase at the margins occurring later in the season. The population decrease in the center can be further attributed to the amplified challenge of finding a mate in a small population, while the population increase at the margins is supported by the dense population observed in that region. To our knowledge, this is the first study that experimentally solves the conundrum of higher damage to fruit in the field margins. We demonstrated a higher population of males and both virgin and mated females at the margins, refuting the possibility of only gravid females migrating into the field. Instead, we suggest the adaptive repellent response to high concentrations of the species-specific sex pheromone as the main cause of the increasing moth populations in the field margins. Declarations Acknowledgments. We thank the farmers from Merom Golan, Sasa, Yron, and Keshet for their cooperation and willingness to contribute their orchards for the research. We thank Hilit Elias and Zeev Farkash for their dedicated technical work. The study was funded by the Israeli Chief Scientist of the Ministry of Agriculture (program No. 21-02-0023 ) and the Plant Council, Israel. Author contributions. Conceptualization: RS and AH. Data curation: RS, MT, AA, and AH. Funding acquisition: RS. Writing & editing: RS, MT, AA, and AH. Data availability. The datasets generated and analyzed during the current study are available from [email protected] upon reasonable request. The authors declare no competing interests. All authors provided their consent to participate and their consent to publish. Ethical Statement: not applicable. References Carde RT and Minks AK, Control of moth pests by mating disruption: successes and constraints. Annu Rev Entomol 40 :559–585 (1995). ‏ Welter S, Pickel C, Millar J, Cave F, Van Steenwyk R and Dunley J, Pheromone mating disruption offers selective management options for key pests. Calif Agric 59 :16–22 (2005). https://doi.org/10.3733/ca.v059n01p16. 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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-4396430","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314919880,"identity":"91962890-2532-4677-affb-033afdddbe77","order_by":0,"name":"Rakefet Sharon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIie3QMQrCMBSA4ScFuxRcC0JzhVcKRUHUo0SEdBIHlw4OcbGLB/ASDi7OkUC7COLmWBF0rZuDiKGCOLUdBfOTJSFfEgKg0/1gPV4HCAEcMNQszddqM1FEUCiyA/RyQt+EVyIDDh8CUEySeJCK8BGsIvOQUeiMSd8ovqW3YBLFDkcbaU1sCqy9FiUP60Iwb97mOaGKSHR5CfEa1+i+fWLgS2t4r0TcJYthy5H60ozzW0jpjx0vQ1vEnqseVm9RZIhGGdkzNxNTh/j75HzMwg6SKDqlReQrC9UJasiK+1Xm+2zCqxOdTqf7j1458FQAr/LqTAAAAABJRU5ErkJggg==","orcid":"","institution":"Northern Research and Development , MIGAL, Institute, Israel","correspondingAuthor":true,"prefix":"","firstName":"Rakefet","middleName":"","lastName":"Sharon","suffix":""},{"id":314919881,"identity":"75f52bb8-7f4a-43cf-a85f-30fc7114bdcd","order_by":1,"name":"Maor Tomer","email":"","orcid":"","institution":"Northern Research and Development , MIGAL, Institute, Israel","correspondingAuthor":false,"prefix":"","firstName":"Maor","middleName":"","lastName":"Tomer","suffix":""},{"id":314919882,"identity":"dccd2c01-af15-4f2f-a772-5ea64051b09f","order_by":2,"name":"Almog Avraham","email":"","orcid":"","institution":"Northern Research and Development , MIGAL, Institute, Israel","correspondingAuthor":false,"prefix":"","firstName":"Almog","middleName":"","lastName":"Avraham","suffix":""},{"id":314919883,"identity":"86a3002c-e797-4f4a-b1a7-487262bf5292","order_by":3,"name":"Ally R Harari","email":"","orcid":"","institution":"Agricultural Research Organization","correspondingAuthor":false,"prefix":"","firstName":"Ally","middleName":"R","lastName":"Harari","suffix":""}],"badges":[],"createdAt":"2024-05-09 16:44:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4396430/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4396430/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58828727,"identity":"72ddb9f2-60a9-41d5-829a-162acf19fb1b","added_by":"auto","created_at":"2024-06-21 17:33:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25387,"visible":true,"origin":"","legend":"\u003cp\u003eInfected fruit (% mean ± SE) on tree canopy at harvest at the margins and center of the orchards. * Paired t-test P\u0026lt;0.05, ** Paired t-test P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4396430/v1/4287440e559dc10c37eb3b52.png"},{"id":58828353,"identity":"24140adc-e3c4-4717-a8b0-3907f54b972e","added_by":"auto","created_at":"2024-06-21 17:33:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50382,"visible":true,"origin":"","legend":"\u003cp\u003eTotal captures in combo traps per day (mean ± SE) at the margins and center of the two orchards. (A) Number of males and females. (B) Percentage of mated females.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4396430/v1/88425e8f1133da32f164bc89.png"},{"id":58828324,"identity":"08b791ac-7ca3-49df-9110-8e5e3f453737","added_by":"auto","created_at":"2024-06-21 17:33:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":223744,"visible":true,"origin":"","legend":"\u003cp\u003eMoth captures in combo traps per day (mean ± SE) at the margins and center of the two orchards: (A, B) Number of males in Merom Golan21 and Keshet, respectively; (C, D) Number of females in Merom Golan21 and Keshet, respectively; (E, F) Percentage of mated females in Merom Golan21 and Keshet, respectively.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4396430/v1/3e484a4206fe34b9ec2931c0.png"},{"id":58944544,"identity":"3507bfbe-e9a3-4d0c-afb9-5ee6a45afcba","added_by":"auto","created_at":"2024-06-24 12:13:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":678789,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4396430/v1/c53fcff2-fd97-4b14-8378-b28465c955f5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Females codling moths evade the mating disruption control tactic","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMating disruption is an environmentally safe pest-control method that employs synthetic sex pheromones to hamper males\u0026apos; ability to locate females. This method is used against various insect pests worldwide, with moths being the predominant target species.\u003csup\u003e1\u0026ndash;7\u003c/sup\u003e The large-scale implementation of pheromone-based mating disruption for pest control offers an opportunity to observe evolution in action.\u003csup\u003e8\u003c/sup\u003e The success of the mating-disruption technique is contingent on both its immediate impact on the targeted pests in the treated area and its sustained, long-term reduction of the pest populations in the region.\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe method is based on preventing encounters of male and female pests. \u0026nbsp;However, both males and females share a common strong interest in reproducing. Consequently, the mating-disruption approach can affect the selection pressures imposed on the sexual communication system, as males and females persist in their efforts to breed. Indeed, although the method has been generally successfully applied for many years and multiple crops, recent observations point to the technique\u0026rsquo;s diminishing efficacy (see Harari and Sharon\u003csup\u003e9\u003c/sup\u003e for review).\u003c/p\u003e\n\u003cp\u003eA local increase in mate encounters may arise from uneven distribution of the synthetic pheromones in a specific field, creating areas with an insufficient dose or no pheromones, where males can readily locate a calling female.\u003csup\u003e1,10\u003c/sup\u003e Uneven distribution of pheromone molecules typically occurs at the margins of pheromone-treated areas.\u003csup\u003e10\u003c/sup\u003e The pheromone concentration in these zones is diminished due to the cascade of its concentration to adjacent areas lacking the mating-disruption pheromones.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnother plausible explanation for the decline of mating-disruption efficacy is the emergence of resistance to the method.\u003csup\u003e9\u003c/sup\u003e For example, after many years of consistent applications of mating disruption in cotton fields to combat the pink bollworm (\u003cem\u003ePectinophora gossypiella\u003c/em\u003e), accumulating evidence suggests that resistance to the method has evolved. In fields treated against this pest, a synthetic pheromone consisting of a blend of the pheromone\u0026rsquo;s two components in a 1:1 ratio had been deployed continually since early 1985.\u003csup\u003e11\u003c/sup\u003e Notably, a substantial number of pink bollworm females in treated fields altered the pheromone ratio, enabling conspecific males to locate their pheromones within the treated areas.\u003csup\u003e12\u003c/sup\u003e Pink bollworm males exposed to the mating-disruption pheromone were able to find these females by recognizing the change in their emitted pheromone. However, they encountered significantly fewer laboratory-reared calling females.\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eSimilarly, following 14 years of continuous applications of mating disruption against the small tea leafroller, \u003cem\u003eAdoxophyes honmai,\u003c/em\u003e in Japan, examination of the pheromone gland in females revealed a threefold increase in the quantity of one of the two pheromone components in females collected from treated as compared to control fields.\u003csup\u003e13\u003c/sup\u003e Importantly, this specific component is for mating disruption. Females may have augmented their pheromone output in an effort to compete with the elevated concentration of this component in the field, aiming to attract males. Nevertheless, the resistance observed in the small tea leafroller was linked to a decline in male sensitivity to this specific pheromone component.\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eA decline in the effectiveness of mating disruption may also be explained by behavioral adaptations aimed at evading pheromone-flooded areas and seeking a pheromone-reduced environment. In such places, females emitting pheromones can successfully attract males. The pheromone-evading behavior implies that females have the ability to detect their species-specific pheromone in their environment.\u003csup\u003e16,17\u003c/sup\u003e Female autodetection was first verified in 1977, when cabbage looper (\u003cem\u003eTrichoplusia ni\u003c/em\u003e) females were caught in female-baited traps.\u003csup\u003e18\u003c/sup\u003e It is evident in their altered behavior in mating disruption-treated fields, as has been documented in various insects, primarily moths. Some moth species exhibit a delay in the onset of calling in the presence of conspecific sex pheromones,\u003csup\u003e19,20\u003c/sup\u003e others demonstrate an earlier calling for males,\u003csup\u003e21,22\u003c/sup\u003e while yet others extend the calling window in an attempt to attract late-coming males.\u003csup\u003e23\u003c/sup\u003e Autodetection of female pheromones also triggers dispersal in certain moth species.\u003csup\u003e21,24,25\u003c/sup\u003e However, in other cases, an excess of female pheromones has also been observed to attract females and increase infestations.\u003csup\u003e26\u003c/sup\u003e The suggestion is that movement away from a concentrated pheromone source, indicative of a high population of competing females, may enhance the likelihood of female mating.\u003csup\u003e10\u003c/sup\u003e This movement could also drive away mated females, thereby reducing resource competition among progeny.\u003csup\u003e27\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThese observations highlight the need for further investigation into the effects of flooding treated fields with species-specific sex pheromones on the dispersal of females, the males\u0026apos; reaction to dispersing females, and the resulting damage to yield.\u003c/p\u003e\n\u003cp\u003eThe commercialization of mating disruption against the codling moth, \u003cem\u003eCydia\u003c/em\u003e \u003cem\u003epomonella\u003c/em\u003e (Tortricidae), was initiated in the United States in 1991.\u003csup\u003e28\u003c/sup\u003e The method is still employed extensively worldwide.\u003csup\u003e29\u003c/sup\u003e In Israel, mating disruption has been used against the codling moth since 1995 and continues to be widely implemented in most apple orchards.\u003csup\u003e30\u003c/sup\u003e This paper tests the hypothesized behavioral changes in response to mating disruption: (i) the virgin females avoid \u0026ldquo;competition\u0026rdquo; by moving to the margin; (ii) the mated females avoid \u0026ldquo;competition\u0026rdquo; on resources by moving to the margin; (iii) males follow the higher population of females at the margin and have better mating success.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSites\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in five commercial apple orchards in northern Israel in 2020\u0026ndash;2021, where the mating-disruption method has been implemented for over 20 years: three plots in the Golan Heights (Merom Golan20, Merom Golan21, and Keshet) and two in the Upper Galilee (Yron and Sasa. The dominant cultivars were Granny Smith and Pink Lady, which were harvested in the autumn (September\u0026ndash;October). We selected plots at the orchards\u0026apos; margins with at least one side that did not share a border with another apple orchard. Mating disruption against the codling moth was applied (CheckMate CM by vapor dispenser; Suterra LLC, Bend, OR, USA) in all orchards at a density of 500 dispensers ha\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor each plot, we defined the margins (2\u0026ndash;3 rows from the edge, ~5\u0026ndash;10 m) and the center (15\u0026ndash;25 rows from the edge, ~100\u0026ndash;150 m). In Keshet, the rows were not parallel to the margin, and the margin was therefore defined as the 2nd to 3rd tree (~5 m), and the center was defined as the 25th tree (100 m).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTreatments and measured variables\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInfected fruit\u003c/p\u003e\n\u003cp\u003eAt apple harvest in 2021 and 2022, we collected 30 random fruits from each of the four trees in each of the orchards\u0026apos; margins and a similar number of fruits from each of the four trees in the center. Fruits were assigned as infected when they had traces of codling moth eggs or exit holes of larvae before pupation. The percentage of infected fruits was calculated for each tree in each orchard\u0026apos;s margin and center. \u0026nbsp;Two-way ANOVA was used to compare the means of infected fruit between the margin and center of the plots, followed by paired t-test for each plot.\u003c/p\u003e\n\u003cp\u003eMonitoring codling moth populations\u003c/p\u003e\n\u003cp\u003eDuring 2021, nine combo traps that attract male and female codling moths (PHEROCON CMDA COMBO + AA, TR\u0026Eacute;C\u0026Eacute;, West Adair, OK, USA) were placed in the margin, and nine traps at the center of each plot. The traps, set in Merom Golan21 in March 2021 and Keshet in May 2021, were checked weekly until the end of May 2022. Collected moths were separated into males and females by their mating organs. Females were dissected to determine their mating status by the presence or absence of at least one full spermatophore in their bursa copulatrix. The percentage of mated females out of all collected female moths was calculated. The number of trapped males and females was compared between each plot\u0026apos;s margin and center by paired t-test. \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eMargin effect on infested fruit\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAn examination of infected fruit during harvest in the apple orchards revealed significantly higher percentages of damaged fruit at the margins than at the center of the plots (two-way ANOVA: F critical = -4.2, DF\u003cem\u003e\u0026nbsp;\u003c/em\u003e= 1, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001). The plots differed in damage level (ANOVA: F critical = -2.7, DF\u003cem\u003e\u0026nbsp;\u003c/em\u003e= 4, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001), with a consistently higher percentage of infected fruit at the margins compared to the center in all orchards (Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMargin effect on codling moth populations\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eComparing the number of males and females captured in the combo traps (Fig. 2A) during the season of 2021 and the spring of 2022 at the margin and center of the plots of two orchards (Merom Golan21 and Keshet) revealed a higher number of trapped males at the margins vs. center of the plots (t-statistic = -1.7, DF = 47, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; and t-statistic = -3.8; DF = 38; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, respectively). The number of trapped\u0026nbsp;\u003c/p\u003e\n\u003cp\u003efemales was also higher in the margins than at the center of the two plots (t-statistic = -3.2, DF = 47, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005; and t-statistic = -2.8, DF = 38, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005, respectively), as were the percentages of mated females in the two plots (Fig. 2B) \u0026nbsp; (t-statistic = -4.4, DF = 35; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; and t-statistic = -2.5, DF = 28, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, respectively).\u003c/p\u003e\n\u003cp\u003eBoth males and females were present at the orchard margins throughout the entire season (April\u0026ndash;September) (Fig. 3). In the center, however, only first-generation males and females were trapped; none were trapped later in the season (Fig. 3A\u0026ndash;D). Mated females followed this pattern (Fig. 3H\u0026ndash;I).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMating disruption, a prevalent agricultural practice, aims to reduce pest populations by manipulating their chemical communication and mating behaviors. Despite its widespread adoption, recent reports from farmers suggest a decline in its effectiveness. This study shows that the diminished efficacy of the method occurs primarily at the orchards\u0026apos; periphery, where the bulk of pest populations and associated damage are concentrated. The findings reveal a notable accumulation of codling moth populations, encompassing both virgin and mated females, males, and offspring, along the apple orchards\u0026apos; margins compared to the orchards\u0026apos; center.\u003c/p\u003e\n\u003cp\u003eAn excess of species-specific pheromones poses mating constraints that challenge both male and female moths to evolve or develop ecological adaptations that will facilitate reproduction. One such adaptive strategy involves altering the pheromone\u0026apos;s characteristics, as evinced in studies with pink bollworm females\u003csup\u003e12\u003c/sup\u003e and the small tea leafroller females.\u003csup\u003e13\u003c/sup\u003e An alternative physiological mechanism to circumvent the effects of excessive pheromones involves adjusting the male\u0026rsquo;s response threshold\u0026mdash;diminishing the response to the component that serves for mating disruption.\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eMinor behavioral adaptations may also enable male moths to evade the disruptive impact of excess pheromones on mate location. For instance, the male codling moth flies upwind, preferentially in the upper reaches of the tree canopy. This strategy allows it to detect females emitting pheromones at the canopy top, which may be above the area in which pheromone dispensers have been distributed.\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eResearch has predominantly focused on the impact of mating-disruption methods on males\u0026apos; ability to locate females,\u003csup\u003e1,31\u003c/sup\u003e and to a lesser extent, the indirect effects on females, such as delayed mating.\u003csup\u003e32\u003c/sup\u003e However, a pheromone-saturated environment may also directly affect the female\u0026apos;s behavior.\u003csup\u003e33\u003c/sup\u003e Documented evidence demonstrates that Lepidoptera and Coleoptera females can autodetect their pheromones.\u003csup\u003e16\u003c/sup\u003e Detecting their species-specific pheromones is advantageous when females compete for breeding opportunities, including mates and resources crucial for their offspring\u0026apos;s survival and fitness.\u003csup\u003e34\u003c/sup\u003e The concentration of species-specific sex pheromones in their immediate surroundings may provide females with information on population size and the intensity of the expected competition.\u003csup\u003e35\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eHence, the flooded pheromone environment resulting from mating disruption may provide a false cue of high population density,\u003csup\u003e36\u003c/sup\u003e implying intense competition for males and potential competition among offspring for resources. This expected competition may change the females\u0026rsquo; dispersal pattern. \u0026nbsp;Indeed, research has revealed a repellent response in both virgin and mated females of the European grape berry moth, \u003cem\u003eLobesia botrana\u003c/em\u003e, when exposed to their own or synthetic versions of their pheromones.\u003csup\u003e37\u003c/sup\u003e This is supported by the behavior of female grape root borers (\u003cem\u003eVitacea polistiformis\u003c/em\u003e) which exhibited a distinct bimodality in vertical distribution when exposed to pheromone treatments, positioning themselves outside the pheromone plume, either higher or lower than control females. By moving, the females exploited the greater air movement and presumably lower pheromone concentration in these areas (Pearson et al., 2004). It has been suggested that moving away from pheromone sources increases the likelihood of female mating success.\u003csup\u003e10,16\u0026nbsp;\u003c/sup\u003eMating disruption may trigger virgin females to move away from areas that are saturated with pheromones,\u0026nbsp;where their ability to be located by mate-seeking males is hindered. Similarly, it may prompt mated females to disperse from regions with high population density, thereby mitigating resource competition among offspring.\u003csup\u003e10,16\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we demonstrate that in apple orchards continuously treated with mating-disruption methods, a significantly higher number of virgin and mated females are found at the orchard margins compared to the center of the orchard. \u0026nbsp;Based on the observed concurrent population reduction in the orchard\u0026apos;s center and its increase at the margins, we suggest that virgin and mated females move to the margins following the cascade in the pheromone concentration, as suggested by Pearson et al.\u0026apos;s model.\u003csup\u003e10\u003c/sup\u003e We also suggest that the higher number of males caught in baited traps in the margins compared to the center results from the males following the calling females to zones with lower pheromone concentrations.\u003c/p\u003e\n\u003cp\u003ePrevious studies of various pest species and crops treated with mating disruption have demonstrated significantly higher damage in the margins. An alternative explanation for this increased damage\u0026nbsp;has been attributed to the invasion of gravid females from nearby untreated hosts.\u003csup\u003e3,4,10,38,39\u0026nbsp;\u003c/sup\u003eHowever, the possibility of gravid females migrating from the fields\u0026apos; outskirts to oviposit in the treated field has scarcely been tested.\u0026nbsp;With the hypothesis of invasion of gravid females in mind, our study was conducted in\u0026nbsp;plots devoid of potential host plants for the codling moth. Furthermore, the presence of a higher number of males at the orchard margins alongside virgin and gravid females does not support the suggested invasion of gravid females as the sole explanation.\u0026nbsp;Rather, our results align more closely with the concept of repellent characteristics associated with high concentrations of species-specific pheromones, as proposed by Pearson et al.\u0026apos;s model\u003csup\u003e10\u003c/sup\u003e and demonstrated by Koutsoumpeli et al.\u003csup\u003e37\u003c/sup\u003e and others.\u003csup\u003e21,24,25\u003c/sup\u003e Accordingly, virgin females move to the margins to reduce the perceived \u0026ldquo;competition\u0026rdquo; for mates, while mated females move to the margins to minimize future \u0026ldquo;competition\u0026rdquo; for their offspring\u0026apos;s resources. Males likely join the higher female population at the margin by following the high concentration of their full-blend pheromone. This is supported by the presence of first-generation male and female moths (April\u0026ndash;May) in both the center and margins of the plots, with the population increase at the margins occurring later in the season. The population decrease in the center can be further attributed to the amplified challenge of finding a mate in a small population, while the population increase at the margins is supported by the dense population observed in that region.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo our knowledge, this is the first study that experimentally solves the conundrum of higher damage to fruit in the field margins. We demonstrated a higher population of males and both virgin and mated females at the margins, refuting the possibility of only gravid females migrating into the field. Instead, we suggest the adaptive repellent response to high concentrations of the species-specific sex pheromone as the main cause of the increasing moth populations in the field margins.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments.\u0026nbsp;\u003c/strong\u003eWe thank the farmers from Merom Golan, Sasa, Yron, and Keshet for their cooperation and willingness to contribute their orchards for the research. We thank Hilit Elias and Zeev Farkash for their dedicated technical work. The study was funded by the Israeli Chief Scientist of the Ministry of Agriculture (program No. 21-02-0023\u003cstrong\u003e)\u0026nbsp;\u003c/strong\u003eand the Plant Council, Israel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions.\u0026nbsp;\u003c/strong\u003eConceptualization: RS and AH. Data curation: RS, MT, AA, and AH. Funding acquisition: RS. \u0026nbsp;Writing \u0026amp; editing: RS, MT, AA, and AH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability.\u0026nbsp;\u003c/strong\u003eThe datasets generated and analyzed during the current study are available from [email protected] \u0026nbsp; upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe authors declare no competing interests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors provided their consent to participate and their consent to publish. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement:\u0026nbsp;\u003c/strong\u003enot applicable.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCarde RT and Minks AK, Control of moth pests by mating disruption: successes and constraints. \u003cem\u003eAnnu Rev Entomol \u003c/em\u003e\u003cstrong\u003e40\u003c/strong\u003e:559\u0026ndash;585 (1995).\u003cspan dir=\"RTL\"\u003e\u0026rlm;\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eWelter S, Pickel C, Millar J, Cave F, Van Steenwyk R and Dunley J, Pheromone mating disruption offers selective management options for key pests. \u003cem\u003eCalif Agric\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e:16\u0026ndash;22 (2005). https://doi.org/10.3733/ca.v059n01p16.\u003c/li\u003e\n\u003cli\u003eHarari AR, Zahavi T, Gordon D, Anshelevich L, Harel M, Ovadia S and Dunkelblum E, Pest management programs in vineyards using male mating disruption. \u003cem\u003ePest Manage Sci\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e:769\u0026ndash;775 (2007). https://doi.org/10.1002/ ps.1365. \u003c/li\u003e\n\u003cli\u003eWitzgall P, Stelinski L, Gut L and Thomson D, Codling moth management and chemical ecology. \u003cem\u003eAnnu Rev Entomol\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e:503\u0026ndash;522 (2008). https://doi.org/10.1146/annurev. ento.53.103106.093323.\u003c/li\u003e\n\u003cli\u003eVacas S, Alfaro C, Navarro-Llopis V and Primo J, Mating disruption of California red scale, \u003cem\u003eAonidiella aurantii\u003c/em\u003e Maskell (Homoptera: Diaspididae), using biodegradable mesoporous pheromone dispensers. \u003cem\u003ePest Manage Sci\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e:745\u0026ndash;751 (2010). https://doi.org/10.1002/ps.1937.\u003c/li\u003e\n\u003cli\u003eLance DR, Leonard DS, Mastro VC and Walters ML, Mating disruption as a suppression tactic in programs targeting regulated lepidopteran pests in US. \u003cem\u003eJ Chem Ecol\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e:590\u0026ndash;605 (2016). https://doi.org/10.1007/ s10886-016-0732-9.\u003c/li\u003e\n\u003cli\u003eSharon R, Zahavi T, Sokolsky T, Sofer-Arad C, Tomer M, Kedoshim R and Harari AR, Mating disruption method against the vine mealybug, \u003cem\u003ePlanococcus ficus\u003c/em\u003e: effect of sequential treatment on infested vines. \u003cem\u003eEntomol Exp Appl\u003c/em\u003e \u003cstrong\u003e161\u003c/strong\u003e:65\u0026ndash;69 (2016). https://doi.org/10.1111/eea.12487.\u003c/li\u003e\n\u003cli\u003eDum\u0026eacute;nil C, Judd GJ, Bosch D, Baldessari M, Gemeno C and Groot AT, Intraspecific variation in female sex pheromone of the codling moth \u003cem\u003eCydia pomonella\u003c/em\u003e. \u003cem\u003eInsects\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e:705\u0026ndash;721\u003cspan dir=\"RTL\"\u003e\u0026rlm;\u003c/span\u003e (2014).\u003c/li\u003e\n\u003cli\u003eHarari A and Sharon R, The contemporary and prospective risks of resistance to the mating disruption method in moths. \u003cem\u003eEntomol Gen\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e:275\u0026ndash;288 (2022).\u003c/li\u003e\n\u003cli\u003ePearson GA, Dillery S and Meyer JR, Modeling intra-sexual competition in a sex pheromone system: how much can female movement affect female mating success? \u003cem\u003eJ Theor Biol\u003c/em\u003e \u003cstrong\u003e231\u003c/strong\u003e:549\u0026ndash;555 (2004).\u003c/li\u003e\n\u003cli\u003eNiv A, Use of pheromones for pink bollworm (\u003cem\u003ePectinophora gossypiella\u003c/em\u003e, Saunders) mating disruption in Israel, in \u003cem\u003eProceedings of the World Cotton Research Conference 2; 1998 Sep 6\u0026ndash;12; Athens, Greece\u003c/em\u003e. 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An increase in successful mate encounters can occur due to uneven distribution of synthetic pheromones in the field, allowing males to locate and mate with calling females in areas of low pheromone concentration and areas with larger pest populations, where random encounters between males and females are more likely to happen by chance.\u003c/p\u003e\n\u003cp\u003eThe study results demonstrate greater fruit damage and higher abundances of male and female codling moths (\u003cem\u003eCydia pomonella)\u003c/em\u003ein the margins compared to the centers of mating disruption-treated apple orchards.\u003c/p\u003e\n\u003cp\u003eWe suggest larger pest populations build up at the orchard margins over the season due to the following behavioral cascade: Females flee areas of high pheromone concentration to reduce the perceived “competition” for mates, while mated females move to the margins to minimize future “competition” for offspring's resources. Males then track the higher female population at the margins by following the females' full pheromone blend.\u003c/p\u003e\n\u003cp\u003eTo our knowledge, this is the first study to offer an interpretation supported by empirical data for the puzzling phenomenon of higher fruit damage in field margins under mating disruption.\u003c/p\u003e","manuscriptTitle":"Females codling moths evade the mating disruption control tactic","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-21 16:19:23","doi":"10.21203/rs.3.rs-4396430/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":"35bc7a8b-1199-442d-a251-ee5516aa1754","owner":[],"postedDate":"June 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-24T12:05:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-21 16:19:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4396430","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4396430","identity":"rs-4396430","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}