{"paper_id":"07897562-2ba8-46f2-ae79-b8cd593a5e72","body_text":"1 \n \nRunning title: Optimal Irradiation Dose for Heterospecific SIT Against Drosophila suzukii 1 \n 2 \nAdvancing Heterospecific Sterile Insect Technique Against 3 \nDrosophila suzukii: selection of the optimal irradiation dose 4 \nfor D. melanogaster males 5 \n 6 \nFlavia Cerasti  1*, Valentina Mastrantonio 1, Alessia Cemmi 2, Ilaria Di Sarcina 2, Massimo 7 \nCristofaro3, Daniele Porretta1 8 \n 9 \n1Department of Environmental Biology, Sapienza University of Rome, Rome, Italy 10 \n2NUC-IRAD-GAM Laboratory, Italian National Agency for New Technologies, Energy and 11 \nSustainable Economic Development (ENEA), Rome, Italy 12 \n3Biotechnology and Biological Control Agency (BBCA), Rome, Italy 13 \n 14 \n* Corresponding author  15 \nE-mail: flavia.cerasti@uniroma1.it (FC) 16 \n 17 \nAbstract 18 \nThe spotted-wing drosophila ( Drosophila suzukii), a highly invasive agricultural pest, poses 19 \nsignificant challenges to fruit production worldwide. Traditional chemical control methods are 20 \ncostly and raise concerns about resistance and environmental sustainability. The Heterospecific 21 \nSterile Insect Technique (h-SIT) has emerged as a promising alternative, using sterile heterospecific 22 \nmales (Drosophila melanogaster ) to suppress D. suzukii  populations through reproductive 23 \ninterference. However, optimizing irradiation doses is critical to balancing male sterility, 24 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n2 \n \nmaintaining biological quality and mating performances. This study aimed to determine the optimal 25 \nirradiation dose for D. melanogaster  males by assessing their sterility, longevity, and courtship 26 \nbehavior following exposure to gamma-ray doses ranging from 80 to 180 Gy. Results showed a 27 \nsignificant reduction in fertility across all irradiation doses, with near-complete sterility at 180 Gy. 28 \nHowever, longevity decreased with increasing doses, with males irradiated at 160–180 Gy showing 29 \na lifespan reduction of up to 50 days compared to controls. Behavioral trials revealed that irradiated 30 \nD. melanogaster males retained their courtship ability toward D. suzukii females, although males 31 \nexposed to 160 Gy exhibited reduced courtship activity. These findings highlight that, among the 32 \ntested doses, 80 Gy emerged as the most effective, preserving male longevity and mating 33 \nperformance while significantly reducing fertility. While 180 Gy achieved the highest sterility, the 34 \npotential lifespan and courtship behavior trade-offs warrant further evaluation. Future studies 35 \nshould evaluate field performance to refine the balance between sterility, longevity, and mating 36 \nperformances for effective D. suzukii population suppression. 37 \n 38 \n1. Introduction 39 \nThe spotted-wing drosophila (SWD), Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is 40 \nan invasive agricultural pest native to Southeast Asia (1). Since its first detection in California in 41 \n2008, D. suzukii has rapidly expanded its geographical distribution in many other states of United 42 \nStates and across the globe, becoming a severe agricultural pest also in Europe, South America, and 43 \nparts of Africa (2–4). According to recent studies, the economic damage caused by D. suzukii in the 44 \nUnited States alone reaches hundreds of millions of US dollars annually, and in Europe, similar 45 \nlosses are reported, suffering significant economic damage in their fruit industries (5,6). The rapid 46 \nspread of D. suzukii has been facilitated by its exceptional ability to thrive in diverse environmental 47 \nconditions, facilitated by its broad temperature tolerance and adaptability to different habitats (1,7). 48 \nOne of the factors contributing to the invasive success of D. suzukii is its nutritional versatility. D. 49 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n3 \n \nsuzukii can attack a broad range of ripening fruits, including soft-skinned berries and stone fruits 50 \nwith economic relevance; then, larval development causes fruit to rot rapidly due to the introduction 51 \nof rot-type pathogens at the oviposition site (8). 52 \nTo counter the negative impact of D. suzukii, efficient and prompt population control actions are 53 \nrequired. Chemical insecticides, especially organophosphates, pyrethroids and spinosyns, were the 54 \nfirst and most effective control approach, but this strategy presents multiple challenges (9,10). 55 \nInsecticides must be applied several times per growing season, due to D. suzukii short generation 56 \ntime and larval development inside the fruits (6,9). Thus, repeated exposure, short generation time, 57 \nand high fecundity have led to metabolic and penetration resistance development to spynosyns and 58 \npyrethroids, raising concerns about the environmental sustainability of this approach (11,12). 59 \nAlternatively, significant research has been devoted to finding sustainable control measures under 60 \nan Integrated Pest Management approach (13–16). 61 \nIn recent years, there has been a renewed interest in the use of SIT (Sterile Insect Technique) 62 \nand the release of sterile heterospecific males (i.e., heterospecific-Sterile Insect Technique) for pest 63 \ncontrol (17–20). Both approaches can be developed under similar theoretical frameworks. SIT 64 \nconsists in releasing large numbers of sterile males of the target pest species into the environment to 65 \nmate with conspecific wild females. The unfertile mating between the released sterile males and 66 \nwild females leads to a gradual decline in the pest population over time (21,22). Contrary to the 67 \nSIT, in heterospecific SIT, sterile males from closely related species are released to compete with 68 \nthe pest population for mates. The heterospecific SIT leverages reproductive interference, a 69 \nreproductive interaction between individuals of different animal co-generic species and/or 70 \nsubspecies, which results in fitness costs for one or both the interacting individuals (23–27). It 71 \nresults from incomplete mating barriers between species and can occur at any stage of mate 72 \nacquisition through different mechanisms, from courtship to mating (24,25,27).  73 \nThe irradiation dose is a key factor for the successful implementation of these approaches, 74 \nrequiring a careful balance between achieving sufficient male sterility and preserving the biological 75 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n4 \n \nperformance of irradiated individuals. Studies on the evaluation of irradiation dose effects on 76 \ndifferent insect pests (e.g., Ceratitis capitata Wiedemann, Anopheles arabiensis Patton, Aedes 77 \naegypti L., Aedes albopictus Skuse) highlighted that an optimal irradiation dose can induce full 78 \nsterility without significantly compromising the biological qualities of males (28–32). At the same 79 \ntime, improperly calibrated irradiation can lead to males that either retain some level of fertility or 80 \nexhibit impaired mate-finding abilities (29,30), emphasizing the necessity of refining the irradiation 81 \ndose to balance sterility and biological fitness. 82 \nOur previous studies demonstrated that Drosophila melanogaster  (Meigen) could be a good 83 \ncandidate for D. suzukii’s control species into a heterospecific SIT context. The two species have 84 \nincomplete pre-mating and complete post-mating isolation, and reproductive interference has been 85 \ndocumented between them (19). Furthermore, under laboratory conditions, D. melanogaster  males 86 \nirradiated at 60 and 80 Gy were able to court and mate with D. suzukii  females, leading to a 87 \nsignificant offspring reduction, although residual fertility has been observed in irradiated males 88 \n(20).  These results provided the first foundation to develop heterospecific SIT against D. suzukii.  89 \nThe aim of this study was to detect the optimal irradiation dose. First, we investigated the effect 90 \nof 6 different doses from 80 to 180 Gy on the sterility of D. melanogaster males and assessed their 91 \nfertility, through mating trials with D. melanogaster females. Second, we investigated the effect of 92 \nthe irradiation on male longevity. Finally, we studied the courtship behavior of irradiated D. 93 \nmelanogaster males analyzing the time spent courting D. suzukii females in relation to the different 94 \nirradiation doses administered. 95 \n 96 \n2. Materials and Methods 97 \n2.1. Fruit fly colonies and rearing techniques 98 \nDrosophila suzukii and D. melanogaster  used in this study were reared at the Sapienza University 99 \nof Rome facilities. The colonies are maintained in the BugDorm-4H4545 insect cages (47.5 x 47.5 x 100 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n5 \n \n47.5 cm) in a thermostatic chamber at 25 ± 1°C, with a 14:10 hour light-dark cycle and with a 101 \nhumidity of 70%. The insects were fed with food substrate based on corn flour (84.1% water, 0.7% 102 \nagar, 3.2% table sugar, 3.6% yeast, 7.2% corn flour, 1.0% soy flour, 0.2% methylparaben dissolved 103 \nin 25 mL of 70% ethanol) (33). Each week, the substrate was replaced with a fresh one to allow the 104 \ninsects to feed and lay eggs. The previous substrate was labeled and placed in specified containers 105 \nto allow the development of new individuals within the colony. The colonies had unrestricted 106 \naccess to water due to cotton balls soaked in a sugar-water solution (1:10 ratio), placed on top of the 107 \ncages. 108 \n 109 \n2.2. Drosophila melanogaster males’ sterilization and individuals’ 110 \nselection 111 \nSterilization of D. melanogaster males was performed at the Calliope gamma irradiation facility at 112 \nthe ENEA Casaccia Research Center (Rome) at different total absorbed doses with a dose rate value 113 \nof about 130 Gy/h. The Calliope is a pool-type facility equipped with n.25 60Co radioisotope 114 \nsources (mean energy 1.25 MeV) in a high volume (7.0 x 6.0  x 3.9 m) shielded cell (34). Males to 115 \nbe sterilized were chosen by checking their emergence from mature pupae into breeding falcons 116 \nevery 30 minutes. In this way, newly emerged males of both species were collected and isolated 117 \nfrom females as soon as they were born, avoiding unwanted mating before the experiment. The 118 \nvirgin males collected were placed in separate cages by species, and after 72-96 hours, they were 119 \ntaken to the irradiation center for sterilization. 120 \n 121 \n2.3. Irradiation effect on D. melanogaster male’ sterility  122 \nTo evaluate the degree of sterility achieved by D. melanogaster  males after irradiation, we 123 \nperformed mating experiments between irradiated D. melanogaster males and fertile D. 124 \nmelanogaster females. Seven treatments were set up, the control using non-irradiated individuals, 125 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n6 \n \nand six treatments relating to the following irradiation doses: 80 Gy, 100 Gy, 120 Gy, 140 Gy, 160 126 \nGy, and 180 Gy. D. melanogaster  males were selected and irradiated as previously described. 127 \nAfterward, we placed five sterilized D. melanogaster  males and five virgin D. melanogaster  128 \nfemales inside 50 ml falcon tubes containing food substrate; we adopted the same procedure for the 129 \ncontrol individuals. We assessed the experiment in a thermostatic chamber at 25 ± 1°C, with a 130 \n14:10 hour light-dark cycle and with a humidity of 70%. After six days, we removed the adult 131 \nindividuals and awaited the emergence of the newborns. We counted and noted newly emerged 132 \nadults daily. We performed five replicates for each condition.  133 \n 134 \n2.4. Irradiation effect on D. melanogaster male’ longevity  135 \nTo evaluate the effect of irradiation on the survival of D. melanogaster  males, we compared the 136 \naverage lifespan between irradiated and non-irradiated D. melanogaster males. We selected 72-96-137 \nhour-old D. melanogaster males that were irradiated as described in the previous paragraph at the 138 \nfollowing doses: 80 Gy, 100 Gy, 120 Gy, 140 Gy, 160 Gy, 180 Gy. For each dose, we set up an 139 \nexperimental cage (30 x 30 x 30 cm) with 20 individuals each. Two other cages were set up as 140 \ncontrols, in which we placed non-irradiated individuals: one cage was called “home control” with 141 \nindividuals maintained at constant conditions of the thermostatic chamber, and one cage called “trip 142 \ncontrol”, with individuals that we transported to the ENEA Calliope facility, but outside of the 143 \nirradiation unit. The “trip control” allowed us to evaluate if the transport could induce an impact on 144 \nthe longevity of the individuals. For all conditions, we carried out mortality checks every day until 145 \nall the individuals died. 146 \n 147 \n2.5. Irradiation effect on D. melanogaster male’ courtship behavior 148 \nWe conducted courtship experiments to evaluate the time spent courting D. suzukii females by D. 149 \nsuzukii and irradiated D. melanogaster  males and to assess potential differences in the courtship 150 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n7 \n \ntime in relation to the administered irradiation doses. We selected individuals following the 151 \nmethods of the previous analyses. In this experiment, we irradiated 72-96 hours old  D. 152 \nmelanogaster males only at 80 Gy, 160 Gy and 180 Gy. We did not consider irradiations at 100, 153 \n120, and 140 Gy because they did not lead to significant differences in terms of sterility and 154 \nlongevity (see Results section).  155 \nWe set up “no-choice” and “choice” experimental trials: in the “no-choice” condition, we 156 \nplaced one D. suzukii  female with homospecific or heterospecific male into a falcon (15 mL) and 157 \nanalyzed the male courtship time. Specifically, we analyzed: - the courtship time of D. suzukii male 158 \nwith a D. suzukii female; - the courtship time of D. melanogaster male irradiated at 80, 160 and 180 159 \nGy with a D. suzukii  female. In the “choice” conditions, we placed one D. suzukii female with two 160 \nhomospecific or heterospecific males into a falcon (15 mL) to evaluate the male's courtship time. 161 \nSpecifically, we analyzed: - the courtship time of two no-irradiated  D. suzukii males with a D. 162 \nsuzukii female; - the courtship time of one no-irradiated D. suzukii male and one D. melanogaster 163 \nmale irradiated at 80, 160 and 180 Gy with a D. suzukii female. For the observation of behaviors 164 \nbetween two D. suzukii males, due to their morphological similarity, the videos were analyzed at 165 \nreduced playback speed to track the individuals and annotate their behaviors accurately. 166 \nConversely, in the second condition involving heterospecific males, the two species were 167 \ndistinguishable: D. suzukii males possess characteristic black spots on their wings (hence the name 168 \n\"spotted-wing drosophila\"), which are absent in D. melanogaster  males. For all conditions, 169 \nfollowing a 5-minute acclimation period, we recorded the individual's behavior for 10 minutes 170 \nusing an Olympus Tough TG-6 camera. After recording, we analyzed the videos using the Boris 171 \nsoftware (Behavioral Observation Research Interactive Software), taking into account the courtship 172 \nelements such as orientation, touch, wing scissoring, wing spreading, and copulation attempt 173 \n(35,36). We carried out 20 replications for each trial to ensure the robustness of our data.  174 \n 175 \n2.6. Data analysis 176 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n8 \n \nIn the sterility experiment, the sterility degree achieved by D. melanogaster  males was 177 \nevaluated for each experimental cond ition, applying a GLM model (generalized linear model with 178 \nnegative binomial distribution), and the model family was selected comparing the AIC and BIC 179 \nestimators and the likelihood ratio test. We performed Tukey's multiple comparison test as a post 180 \nhoc test using the ‘multcomp’  package (37).  The average percentage of residual fertility in each 181 \ncondition was obtained by calculating the percentage reduction of each replicate in a specific 182 \ncondition compared to the mean of offspring born in the control condition (i.e. 100% fertility) and 183 \ncalculating the mean (± SE) of the percentages obtained in each replicate. 184 \nTo evaluate the effect of the irradiation dose on the longevity of D. melanogaster  males, 185 \nsurvival distributions of the different D. melanogaster groups (‘Control cages’, ‘Control trip’, ‘80 186 \nGy’, ‘100 Gy’, ‘120 Gy’, ‘140 Gy’, ‘160 Gy’, ‘180 Gy’) were computed using the Kaplan-Meier 187 \nmethod with the ‘ survival’ package and the differences between survival distributions were 188 \nestimated using the Log-Rank Test with the ‘survminer’ package (38,39).    189 \nIn the courtship experiment, to compare the courtship time of D. suzukii and D. melanogaster  190 \nmales in “no-choice” condition, we used a GLM model (generalized linear model with negative 191 \nbinomial distribution), selecting the model family based on the AIC and BIC estimators and the 192 \nlikelihood ratio test.  We performed Tukey's multiple comparison test as a post hoc test. In the 193 \n“choice” condition, we used a GLM model (generalized linear model with negative binomial 194 \ndistribution) to compare the average courtship time of males. Then, we compared the average 195 \ncourtship time of the two males in the same condition using the nonparametric statistical Wilcoxon 196 \nSigned Rank test using the ‘dplyr’ package (40). All analyses were carried out using R Software 197 \nversion 3.6.2. (41). 198 \n 199 \n3. Results 200 \n3.1. Drosophila melanogaster males’ sterilization  201 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n9 \n \nWe assessed the sterility of D. melanogaster males by mating them with D. melanogaster females 202 \nat irradiation doses of 80, 100, 120, 160, and 180 Gy, with a non-irradiated control group for 203 \ncomparison. The mean number of the emerged adults in the control condition was 171.8 (± 24.47) 204 \n(mean ± SE) (Table 1). A significant emergence reduction was observed at all irradiation doses. At 205 \nthe 80 Gy irradiation condition, the mean number of emerged adults was  29.2 (± 9.25), while at 206 \n180 Gy irradiation, it dropped to 0.8 (± 0.58) (Table 1; Fig 1). The GLM model showed a 207 \nsignificant effect of the male irradiation dose on the number of offspring produced by D. 208 \nmelanogaster females (Table 2). Tukey's multiple comparison test showed a significant offspring 209 \nreduction from the control condition to all irradiation conditions, i.e. 80 Gy (z = 4.253, p = <0.001), 210 \n100 Gy (z = 5.183, p = <0.001), 120 Gy (z = 5.634, p = <0.001), 140 Gy (z = 6.660, p = <0.001), 211 \n160 Gy (z = 7.194, p = <0.001) and 180 Gy (z = 8.317, p = <0.001). There were also significant 212 \ndifferences between the 80 Gy irradiation dose and 160 Gy (z = 3.497, p= 0.008) and 180 Gy (z = 213 \n5.535, p= <0.001) doses. The 180 Gy dose showed significant differences with 80 Gy (see above), 214 \n100 Gy (z = -4.886, p= <0.001), 120 Gy (z = -4.559, p= <0.001) and 140 Gy (z = -3.761, p= 0.003) 215 \ndoses, but not with 160 Gy. We did not observe significant differences between 80, 100, 120 and 216 \n140 Gy (Fig 1). 217 \n 218 \nTable 1 . The mean number of the emerged D. melanogaster  219 \nadults. Mean number (±SE) of emerged adults and average 220 \npercentage (±SE) of the residual fertility at the different treatment 221 \ndoses (Gy). 222 \nTreatment Dose (Gy) \nMean number of \nemerged adults \n(±SE) \nAverage percentage \n(±SE) of residual \nfertility  \n0 Gy 171.8 (± 24.47) 100 % \n80 Gy 29.2 (± 9.25)  17 % (± 5.38) \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n10 \n \n100 Gy 23.5 (± 5.52)  11.29 % (± 3.45) \n120 Gy 19.5 (± 2.25)  9.19 % (± 2.38) \n140 Gy 11.75 (± 2.66)  5.59 % (± 1.73) \n160 Gy 5.25 (± 2.39)  3.05 % (± 24.47) \n180 Gy 0.8 (± 0.58)  0.47 % (± 0.34) \n 223 \n 224 \nTable 2. Irradiation effect on D. melanogaster  male’ sterility.  GLM model values are shown. 225 \nValues in boldface indicate significant differences. 226 \nFixed Effects Estimate ±SE z Value p-Value \n     \n(Intercept) 2.9653 0.3051 9,718 < 2e-16 \n120 Gy -0.2053 0.4342 -0.473 0.63643 \n140 Gy -0.7035 0.4435 -1.586 0.11272 \n160 Gy -1.3070 0.4942 -2.645 0.00817 \n180 Gy -3.1884 0.6526 -4.886 1..03e-06 \n80 Gy 0.4089 0.4275 0.956 0.33882 \nNo irradiation 2.1811 0.4208 5.183 2.18e-07 \n 227 \n 228 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n229 \nFig 1.  Irradiation effect on D. melanogaster  male’ sterility.  The number of D. melanogaster  adults 230 \nemerged from fertile females and irradiated males. Different letters mean significant differences by Tukey 231 \nMultiple Comparison tests (p < 0.05). 232 \n 233 \n3.2. Irradiation effects on D. melanogaster male’s longevity 234 \nIn the longevity tests of D. melanogaster  males, Kaplan- Meier curves showed significant 235 \ndifferences in lifespan between the treatments (80 Gy, 100 Gy, 120 Gy, 140 Gy, 160 Gy, 180 Gy, 236 \nthe ‘home control and ‘trip control’ conditions) (Mantel-Cox log-rank; χ 2 = 105.9, d.f.= 7, P = < 2e-237 \n16) (Fig 2). The pairwise comparison test showed that control individuals have a higher probability 238 \nof survival than irradiated individuals. In particular, the ‘trip control’ condition showed significant 239 \ndifferences with all the irradiation doses tested (80 Gy, p = 0.0 39; 100 Gy, p = 0.027; 120 Gy, p = 240 \n0.013; 140 Gy, p = 0.017; 160 Gy, p < 0.001; 180 Gy, p < 0.001; ‘home control’ condition, p = 241 \n0.014), with an average life of 71 days. Instead, the ‘home control’ condition showed significant 242 \ndifferences only with 160 Gy (p = 0.001), 180 Gy (p = 0.027), and the ‘trip control’ condition, with 243 \nan average life of 68 days. Significant differences were also observed in the longevity of males 244 \nirradiated at 80 Gy and those irradiated at 160 Gy (p < 0.001), 180 Gy (p < 0.001), and the ‘trip 245 \n \nlts \ney \nnt \ny, \n-\nity \nnt \n = \n = \nnt \nith \nes \nip \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\ncontrol’ condition. The longevity of individuals irradiated at 160 Gy is significantly different from 246 \nboth control treatments and 80 Gy irradiation and also from 100 Gy (p < 0.001), 120 Gy ( p < 247 \n0.001), 140 Gy (p < 0.001) and 180 Gy (p = 0.013).  Furthermore, we found that the longevity of 248 \nindividuals irradiated at 180 Gy is significantly different from both control treatments, 80 Gy and 249 \n160 Gy, and also from 100 Gy (p < 0.001), 120 Gy (p < 0.001), and 140 Gy (p < 0.001 ). The 250 \naverage life of individuals irradiated at 160 Gy was 46 days and irradiated at 180 Gy was 55 days. 251 \nWe found no significant differences between the dose irradiations 80, 100, 120 and 140 Gy, with an 252 \naverage life of 63 days (Fig 2). 253 \n 254 \nFig 2. Irradiation effect on D. melanogaster male longevity.  Kaplan-Meier curves showing the effect of 255 \ndifferent irradiation doses on the longevity of D. melanogaster males. 256 \n 257 \n3.3. Irradiation effect on D. melanogaster male’ courtship behavior 258 \nIn the “no-choice” conditions, the mean (± SE) courtship time widely ranged from 16.70% (± 3.98) 259 \n(D. melanogaster irradiated at 160 Gy) up to 67.66% (± 8.30) (D. suzukii males) (Fig 3). The GLM 260 \nmodel showed significant differences in the average courtship time among conditions (Table 3). 261 \nTukey's multiple comparison tests showed a significant difference between D. suzukii homospecific 262 \nm \n < \nof \nnd \nhe \ns. \nan \nof \n8) \nM \n). \nfic \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n13 \n \ncondition and the conditions with D. melanogaster irradiated at 160 Gy (z = 4.346, p = <0.001) and 263 \n180 Gy (z = 3.552, p = 0.002) and also a significant difference between D. melanogaster irradiated 264 \nat 80 Gy and 160 Gy (z = 2.697, p = 0.035). No significant differences were observed in terms of 265 \ncourtship time between D. suzukii males and D. melanogaster males irradiated at 80 Gy (p > 0.05) 266 \nand between the courtship time of D. melanogaster irradiated at 80 and 180 Gy (p > 0.05) (Fig 3).  267 \nIn the “choice” trials, the mean courtship times varied across treatments. In the conspecific 268 \ncondition with two D. suzukii males, the mean courtship time was 24.23% (± 6.17) for one male and 269 \n16% (± 3.50) for the other. In the heterospecific condition with irradiated D. melanogaster males at 270 \n80 Gy, the D. suzukii  male exhibited a mean courtship time of 12.60% (± 3.06), while the D. 271 \nmelanogaster male displayed 31.83% (± 7.37). In the heterospecific condition with irradiated D. 272 \nmelanogaster males at 160 Gy, the D. suzukii male showed a mean courtship time of 24% (± 4.88), 273 \nwhereas the D. melanogaster male had 9.66% (± 4.61). In the heterospecific condition with D. 274 \nmelanogaster males irradiated at 180 Gy, the courtship time was 12.59% (± 1.94) for the D. suzukii 275 \nmale and 13.58% (± 5.68) for the D. melanogaster male (Fig 4).  276 \nThe GLM analysis revealed significant differences in the average courtship time among conditions 277 \n(Table 4), and Tukey’s multiple comparisons test indicated a significant difference only between the 278 \ncourtship times of irradiated D. melanogaster males irradiated at 80 Gy and 160 Gy (p = 0.016; Fig 279 \n4). The Wilcoxon rank sum test highlighted significant differences only in the total courtship time 280 \nbetween D. melanogaster males irradiated at 160 Gy and D. suzukii  males (W = 82.5, p = 0.002) 281 \n(Fig 4).  282 \n 283 \nTable 3. Irradiation effect on D. melanogaster  male’ courtship time in “no-choice” 284 \ncondition. GLM model values are shown. Values in boldface indicate significant differences. D. 285 \nsuz = Drosophila suzukii; D. mel = Drosophila melanogaster. 286 \nFixed Effects Estimate ±SE z Value p-Value \n     \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n14 \n \n(Intercept) 2.8151 0.2273 12,384 < 2e-16 \nD. mel 180 Gy 0.2592 0.3204 0.809 0.41853 \nD. mel 80 Gy 0.8595 0.3188 2.697 0.00069 \nD. suz  \n(homospecific condition) 1.3994 0.3220 4.346 1.39e-05 \n 287 \n 288 \nTable 4. Irradiation effect on D. melanogaster  male’ courtship time in “choice” condition. 289 \nGLM model values are shown. Values in boldface indicate significant differences. D. suz  = 290 \nDrosophila suzukii; D. mel = Drosophila melanogaster. 291 \nFixed Effects Estimate ±SE z Value p-Value \n     \n(Intercept) 3.4604 0.2447 14,139 < 2e-16 \nD. suz 80 Gy -0.9271 0.3496 -2.652 0.00799 \nD. mel 160 Gy -1.1924 0.3513 -3.394 0.00068 \nD. suz 160 Gy -0.2828 0.3469 -0.815 0.41493 \nD. mel 180 Gy -0.8515 0.3538 -2.407 0.01609 \nD. suz 180 Gy -0.9279 0.3496 -2.654 0.00794 \nD. suz 1  \n(homospecific condition) -0.6934 0.3530 -1.964 0.04948 \nD. suz 2 \n(homospecific condition) -0.2730 0.3514 -0.777 0.43724 \n 292 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n 293 \nFig 3. Courtship comparison in the “no-choice” trials.  Courtship of D. suzukii males toward D. suzukii294 \nfemales (pink column); courtship of D. melanogaster males toward D. suzukii females at different irradiation 295 \ndoses (blue, green and orange columns). *** Tukey's multiple com- parison tests p < 0.001; ** Tukey's 296 \nmultiple comparison tests p < 0.01; * Tukey's multiple comparison tests p-value < 0.05. Black dots are box -297 \nplot outliers. 298 \n 299 \nkii \non \ny's \n-\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n300 \nFig 4.  Courtship comparisons in “choice” trials.   Time spent courting D. suzukii  females by D. suzu kii301 \nmales and irradiated D. melanogaster  males at 80, 160 and 180 Gy. D. suz  = D. suzukii  males (pink 302 \ncolumns); D. mel 80 Gy = D. melanogaster  males irradiated at 80 grey (blue column); D. mel 160 Gy = D. 303 \nmelanogaster males irradiated at 160 grey (green column); D. mel  180 Gy = D. melanogaster  males 304 \nirradiated at 180 grey (orange column).   Black dots are box-plot outliers. * Tukey's multiple comparison 305 \ntests p-value < 0.05; ** Wilcoxon rank sum test p < 0.01.  306 \n 307 \n4. Discussion 308 \nFinding the best irradiation dose is a crucial issue that requires careful evaluation to develop a 309 \nheterospecific SIT approach. We found that irradiation was highly effective at reducing fertility. All 310 \nirradiation doses led to a significant reduction in adult emergence with respect to the control 311 \ncondition (Table 1; Fig 1). We observed at the lower irradiation dose tested (80 Gy), only a 17% (± 312 \n5.38) average residual fertility that decreases as the irradiation doses increase until reaching 0.47 (± 313 \n0.34) average residual fertility at the highest dose tested (180 Gy) (Table 1). These results are 314 \nconsistent with previous findings. Studies about the effect of gamma rays on the sterility of D. 315 \n \nkii \nnk \nD. \nes \non \n a \nll \nol \n(± \n(± \nre \nD. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n17 \n \nmelanogaster have been carried out in the 1960s and ‘70s. Henneberry (1963) (42) found that D. 316 \nmelanogaster irradiated males at 160 Gy dose produced nonviable eggs after mating with non-317 \nirradiated females. Accordingly, Nelson (1973) (43) observed that at 120 Gy 99.3% fewer progeny 318 \nemerged than non-irradiated individuals. 319 \nA trade-off between the sterility and longevity of the irradiated males is critical in 320 \noptimizing classic and heterospecific SIT applications (44). The survival analysis showed that 321 \nirradiation significantly reduces the lifespan of D. melanogaster  males, with the highest reductions 322 \nin longevity at the highest doses. Control individuals lived on average for 70 days, whereas males 323 \nirradiated at the highest doses (160–180 Gy) experienced a 50-day lifespan (Fig 2). This 324 \nobservation aligns with prior studies indicating that irradiation-induced oxidative stress and cellular 325 \ndamage can impair physiological functions, shortening lifespan (45). Nelson et al. (1973) (43) also 326 \nreported decreased longevity in irradiated D. melanogaster, with a similar reduction in lifespan at 327 \nthe highest dose tested of 150 Gy. The dose-dependent decrease in longevity must be carefully 328 \nconsidered when applying SIT since male competitiveness may be compromised if they do not 329 \nsurvive long enough to mate effectively in the wild. A reduction in the average lifespan of D. 330 \nmelanogaster from 70 in the controls to 50 days at the highest radiation doses can be seen unlike to 331 \ncompromise the effectiveness of SIT, as frequent releases of sterile individuals are typically part of 332 \nthe strategy. For instance, regarding screwworm Cochliomyia hominivorax (Coquerel), the releases 333 \nhave to occur weekly to maintain the critical ratio or even twice a week for the Mediterranean fruit 334 \nfly and tsetse Glossina austeni  (Wiedemann) (46–48). We want to highlight that this study was not 335 \naddressed to assess the longevity of sterile individuals in field conditions, which can be lower than 336 \nin protected field-cage situations, where sterile males have easy access to food and are protected 337 \nfrom predation in the laboratory (49). This aspect certainly warrants further investigation. 338 \nThe last part of this study was designed to assess the heterospecific courtship behavior of 339 \nirradiated D. melanogaster  males. A balance between sterility and behavioral competence when 340 \nselecting an irradiation dose for pest control is critical. If males lose the ability to court females, 341 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n18 \n \ntheir sterility will have limited impact on population suppression, as seen in studies of other insect 342 \nspecies, such as the Queensland fruit fly ( Bactrocera tryoni Froggatt), where high doses impaired 343 \nsexual competitiveness (50). Our findings showed that irradiated D. melanogaster males retained 344 \ntheir ability to court D. suzukii  females even at the highest irradiation doses, suggesting that 345 \ncourtship behavior remains largely unaffected by irradiation. In the “no-choice condition”, however, 346 \nD. melanogaster males irradiated at 160 and 180 Gy exhibited significantly lower courtship activity 347 \ncompared to D. suzukii  males toward conspecific females (Table 3; Fig 3).  Conversely, under the 348 \n“choice condition”, D. melanogaster males courted D. suzukii females as much as D. suzukii males, 349 \neven at the highest irradiation doses tested. Notably, D. melanogaster  males irradiated at 160 Gy 350 \nshowed reduced courtship toward D. suzukii females compared to D. melanogaster males irradiated 351 \nat 80 Gy and D. suzukii males, corroborating the observations made in the “no-choice condition” 352 \n(Fig 4). These results suggest two key points. First, the presence of D. melanogaster males seems to 353 \ninfluence the courtship behavior of D. suzukii males, as they courted conspecific females more in 354 \nthe “no-choice condition” compared to the “choice condition”. The reduced courtship behavior 355 \nobserved at a radiation dose of 160 Gy suggests that higher doses may lead to behavioral 356 \nimpairments in D. melanogaster males. These impairments are likely attributable to physiological 357 \nalterations or disruptions in neural circuits essential for mating displays. Ionizing radiation is known 358 \nto damage neural pathways involved in courtship behavior, as evidenced in moths, where higher 359 \ndoses often result in physiological defects that reduce their competitiveness with wild populations 360 \n(51,52). Radiation may also interfere with producing or expressing key biochemical and behavioral 361 \nsignals. During courtship, D. melanogaster  males emit specific biochemical signals, such as cis-362 \nvaccenyl acetate, along with behavioral signals like wing vibrations and pheromone release, to 363 \nstimulate female responses (53–55). Ionizing radiation may disrupt these signals, compromising the 364 \nmale's ability to communicate with females effectively. Similar disruptions have been observed in 365 \nother pest species, such as Callosobruchus chinensis L. females and Anthonomus grandis  366 \n(Boheman) males, where radiation-induced impairments in mating signals led to reduced courtship 367 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n19 \n \nand mating success (56,57). Consequently, irradiated D. melanogaster  males may fail to elicit 368 \nappropriate responses from females, disrupting courtship and mating dynamics. In our study, while 369 \nD. melanogaster males irradiated at 160 Gy showed reduced courtship, males exposed to 180 Gy 370 \ncourted D. suzukii females comparably to untreated D. suzukii  males under both \"no-choice\" and 371 \n\"choice\" conditions (Figs 3 and 4). Additionally, the highest courtship percentage was observed at 372 \nthe lowest tested dose of 80 Gy (Figs 3 and 4), supporting the notion that lower radiation doses may 373 \npreserve male courtship behavior more effectively. 374 \nOverall, our study highlights the complex interactions between irradiation, longevity, 375 \nsterility, and mating behavior in D. melanogaster and contributes to growing evidence of using 376 \nheterospecific SIT in pest control. Based on our results, the 80 Gy and 180 Gy radiation doses 377 \nappear most suitable for further investigation. At 80 Gy, we observed the highest courtship rates 378 \nand longest lifespan among the radiation doses tested. In comparison, at 180 Gy, we achieved the 379 \ngreatest reduction in fertility. 380 \nFor an effective SIT program, it is essential that sterile males survive for a long time in their 381 \nenvironment. Only in this way they can mate with a sufficient number of wild females and induce 382 \nsterility in the population. If their quality is compromised and their longevity reduced, more 383 \nfrequent and larger-scale releases will be required to sustain a high overflooding ratio, ultimately 384 \nincreasing operational costs (58). Studies on An. arabiensis  and Ae. aegypti  have highlighted 385 \ndifferent factors influencing longevity. An. arabiensis was found to have a significantly shorter 386 \nlifespan in field settings compared to laboratory conditions, whereas Ae. aegypti  exhibited a 387 \nstronger sensitivity to seasonality (59). Specifically, in a field experiment carried out in Vietnam, 388 \nAe. aegypti populations demonstrated significantly higher survival rates during the cool or hot dry 389 \nseasons compared to the cool and wet seasons (60). Moreover, a synergistic effect of irradiation, 390 \npacking, and chilling was observed to compromise the longevity of An. arabiensis —an effect that 391 \nwas not detected in Ae. aegypti (59). These results suggest that the impact of irradiation on lifespan 392 \nis highly species-dependent. In our case, it is essential first to assess the differences in D. 393 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n20 \n \nmelanogaster males' longevity kept under laboratory conditions and those exposed to environmental 394 \nconditions. This would allow us to better evaluate the lifespan reduction at 180 Gy and determine 395 \nwhether it constitutes a limitation for SIT applications. Conversely, a higher residual fertility at 80 396 \nGy than 180 Gy might be less restrictive given that we are not dealing with a pest or invasive 397 \nspecies. Unlike in standard SIT applications for pest control, achieving the high sterility levels 398 \nrequired to release pest males (as discussed by Bakri et al. 2021 (61) and Parker et al. 2021 (62)) 399 \nmay not be necessary in this context. Based on these considerations, the present study has 400 \ndemonstrated that an irradiation dose of 80 Gy seems to be more effective. However, further studies 401 \nare needed better to evaluate both longevity and mating performance under field conditions. 402 \nGreenhouses and other enclosed environments appear ideal for implementing the 403 \nheterospecific Sterile Insect Technique (h-SIT) to manage D. suzukii populations. Studies on 404 \nplastic- and mesh-covered tunnels have shown that mechanical barriers alone can significantly 405 \nreduce D. suzukii populations in these confined areas. This reduction is due not only to the physical 406 \nexclusion provided by the barriers but also to creating an unfavorable microclimate for the pest's 407 \nsurvival (63). Although complete exclusion cannot be achieved through mechanical barriers alone, 408 \nintegrating h-SIT with these measures could enhance the overall effectiveness of biocontrol 409 \nstrategies.  410 \n 411 \nConclusions 412 \nOur findings highlight the critical balance between sterility, longevity, and mating behavior in D. 413 \nmelanogaster for heterospecific SIT applications. Among the tested doses, 80 Gy emerged as the 414 \nmost effective, preserving male longevity and mating performance while significantly reducing 415 \nfertility. While 180 Gy achieved the highest sterility, the potential lifespan and courtship behavior 416 \ntrade-offs warrant further evaluation. Future studies should focus on-field performance to refine SIT 417 \nprotocols. Integrating h-SIT with mechanical barriers in controlled environments like greenhouses 418 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint \n\n21 \n \ncould enhance D. suzukii  management, making 80 Gy a promising dose for practical 419 \nimplementation. 420 \n 421 \nAcknowledgments 422 \nWe thank Alessandra Spanò, Elisa Michelangeli and Giulia Pezzi for their technical help.  423 \n 424 \n 425 \n 426 \n 427 \nReferences 428 \n1. Cini A, Ioriatti C, Anfora G. A review of the invasion of Drosophila suzukii  in Europe and a 429 \ndraft research agenda for integrated pest management. 2012 [cited 2025 Jan 21]; Available 430 \nfrom: https://openpub.fmach.it/handle/10449/21029 431 \n2. 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