Advancing Heterospecific Sterile Insect Technique Against Drosophila suzukii:selection of the optimal irradiation dose for D. melanogaster males

18 The spotted-wing drosophila ( Drosophila suzukii), a highly invasive agricultural pest, poses 19 significant challenges to fruit production worldwide. Traditional chemical control methods are 20 costly and raise concerns about resistance and environmental sustainability. The Heterospecific 21 Sterile Insect Technique (h-SIT) has emerged as a promising alternative, using sterile heterospecific 22 males (Drosophila melanogaster ) to suppress D. suzukii populations through reproductive 23 interference. However, optimizing irradiation doses is critical to balancing male sterility, 24 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 2 maintaining biological quality and mating performances. This study aimed to determine the optimal 25 irradiation dose for D. melanogaster males by assessing their sterility, longevity, and courtship 26 behavior following exposure to gamma-ray doses ranging from 80 to 180 Gy. Results showed a 27 significant reduction in fertility across all irradiation doses, with near-complete sterility at 180 Gy. 28 However, longevity decreased with increasing doses, with males irradiated at 160–180 Gy showing 29 a lifespan reduction of up to 50 days compared to controls. Behavioral trials revealed that irradiated 30 D. melanogaster males retained their courtship ability toward D. suzukii females, although males 31 exposed to 160 Gy exhibited reduced courtship activity. These findings highlight that, among the 32 tested doses, 80 Gy emerged as the most effective, preserving male longevity and mating 33 performance while significantly reducing fertility. While 180 Gy achieved the highest sterility, the 34 potential lifespan and courtship behavior trade-offs warrant further evaluation. Future studies 35 should evaluate field performance to refine the balance between sterility, longevity, and mating 36 performances for effective D. suzukii population suppression. 37 38 1. Introduction 39 The spotted-wing drosophila (SWD), Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is 40 an invasive agricultural pest native to Southeast Asia (1). Since its first detection in California in 41 2008, D. suzukii has rapidly expanded its geographical distribution in many other states of United 42 States and across the globe, becoming a severe agricultural pest also in Europe, South America, and 43 parts of Africa (2–4). According to recent studies, the economic damage caused by D. suzukii in the 44 United States alone reaches hundreds of millions of US dollars annually, and in Europe, similar 45 losses are reported, suffering significant economic damage in their fruit industries (5,6). The rapid 46 spread of D. suzukii has been facilitated by its exceptional ability to thrive in diverse environmental 47 conditions, facilitated by its broad temperature tolerance and adaptability to different habitats (1,7). 48 One of the factors contributing to the invasive success of D. suzukii is its nutritional versatility. D. 49 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 3 suzukii can attack a broad range of ripening fruits, including soft-skinned berries and stone fruits 50 with economic relevance; then, larval development causes fruit to rot rapidly due to the introduction 51 of rot-type pathogens at the oviposition site (8). 52 To counter the negative impact of D. suzukii, efficient and prompt population control actions are 53 required. Chemical insecticides, especially organophosphates, pyrethroids and spinosyns, were the 54 first and most effective control approach, but this strategy presents multiple challenges (9,10). 55 Insecticides must be applied several times per growing season, due to D. suzukii short generation 56 time and larval development inside the fruits (6,9). Thus, repeated exposure, short generation time, 57 and high fecundity have led to metabolic and penetration resistance development to spynosyns and 58 pyrethroids, raising concerns about the environmental sustainability of this approach (11,12). 59 Alternatively, significant research has been devoted to finding sustainable control measures under 60 an Integrated Pest Management approach (13–16). 61 In recent years, there has been a renewed interest in the use of SIT (Sterile Insect Technique) 62 and the release of sterile heterospecific males (i.e., heterospecific-Sterile Insect Technique) for pest 63 control (17–20). Both approaches can be developed under similar theoretical frameworks. SIT 64 consists in releasing large numbers of sterile males of the target pest species into the environment to 65 mate with conspecific wild females. The unfertile mating between the released sterile males and 66 wild females leads to a gradual decline in the pest population over time (21,22). Contrary to the 67 SIT, in heterospecific SIT, sterile males from closely related species are released to compete with 68 the pest population for mates. The heterospecific SIT leverages reproductive interference, a 69 reproductive interaction between individuals of different animal co-generic species and/or 70 subspecies, which results in fitness costs for one or both the interacting individuals (23–27). It 71

from incomplete mating barriers between species and can occur at any stage of mate 72 acquisition through different mechanisms, from courtship to mating (24,25,27). 73 The irradiation dose is a key factor for the successful implementation of these approaches, 74 requiring a careful balance between achieving sufficient male sterility and preserving the biological 75 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 4 performance of irradiated individuals. Studies on the evaluation of irradiation dose effects on 76 different insect pests (e.g., Ceratitis capitata Wiedemann, Anopheles arabiensis Patton, Aedes 77 aegypti L., Aedes albopictus Skuse) highlighted that an optimal irradiation dose can induce full 78 sterility without significantly compromising the biological qualities of males (28–32). At the same 79 time, improperly calibrated irradiation can lead to males that either retain some level of fertility or 80 exhibit impaired mate-finding abilities (29,30), emphasizing the necessity of refining the irradiation 81 dose to balance sterility and biological fitness. 82 Our previous studies demonstrated that Drosophila melanogaster (Meigen) could be a good 83 candidate for D. suzukii’s control species into a heterospecific SIT context. The two species have 84 incomplete pre-mating and complete post-mating isolation, and reproductive interference has been 85 documented between them (19). Furthermore, under laboratory conditions, D. melanogaster males 86 irradiated at 60 and 80 Gy were able to court and mate with D. suzukii females, leading to a 87 significant offspring reduction, although residual fertility has been observed in irradiated males 88 (20). These results provided the first foundation to develop heterospecific SIT against D. suzukii. 89 The aim of this study was to detect the optimal irradiation dose. First, we investigated the effect 90 of 6 different doses from 80 to 180 Gy on the sterility of D. melanogaster males and assessed their 91 fertility, through mating trials with D. melanogaster females. Second, we investigated the effect of 92 the irradiation on male longevity. Finally, we studied the courtship behavior of irradiated D. 93 melanogaster males analyzing the time spent courting D. suzukii females in relation to the different 94 irradiation doses administered. 95 96 2. Materials and Methods 97 2.1. Fruit fly colonies and rearing techniques 98 Drosophila suzukii and D. melanogaster used in this study were reared at the Sapienza University 99 of Rome facilities. The colonies are maintained in the BugDorm-4H4545 insect cages (47.5 x 47.5 x 100 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 5 47.5 cm) in a thermostatic chamber at 25 ± 1°C, with a 14:10 hour light-dark cycle and with a 101 humidity of 70%. The insects were fed with food substrate based on corn flour (84.1% water, 0.7% 102 agar, 3.2% table sugar, 3.6% yeast, 7.2% corn flour, 1.0% soy flour, 0.2% methylparaben dissolved 103 in 25 mL of 70% ethanol) (33). Each week, the substrate was replaced with a fresh one to allow the 104 insects to feed and lay eggs. The previous substrate was labeled and placed in specified containers 105 to allow the development of new individuals within the colony. The colonies had unrestricted 106 access to water due to cotton balls soaked in a sugar-water solution (1:10 ratio), placed on top of the 107 cages. 108 109 2.2. Drosophila melanogaster males’ sterilization and individuals’ 110 selection 111 Sterilization of D. melanogaster males was performed at the Calliope gamma irradiation facility at 112 the ENEA Casaccia Research Center (Rome) at different total absorbed doses with a dose rate value 113 of about 130 Gy/h. The Calliope is a pool-type facility equipped with n.25 60Co radioisotope 114 sources (mean energy 1.25 MeV) in a high volume (7.0 x 6.0 x 3.9 m) shielded cell (34). Males to 115 be sterilized were chosen by checking their emergence from mature pupae into breeding falcons 116 every 30 minutes. In this way, newly emerged males of both species were collected and isolated 117 from females as soon as they were born, avoiding unwanted mating before the experiment. The 118 virgin males collected were placed in separate cages by species, and after 72-96 hours, they were 119 taken to the irradiation center for sterilization. 120 121 2.3. Irradiation effect on D. melanogaster male’ sterility 122 To evaluate the degree of sterility achieved by D. melanogaster males after irradiation, we 123 performed mating experiments between irradiated D. melanogaster males and fertile D. 124 melanogaster females. Seven treatments were set up, the control using non-irradiated individuals, 125 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 6 and six treatments relating to the following irradiation doses: 80 Gy, 100 Gy, 120 Gy, 140 Gy, 160 126 Gy, and 180 Gy. D. melanogaster males were selected and irradiated as previously described. 127 Afterward, we placed five sterilized D. melanogaster males and five virgin D. melanogaster 128 females inside 50 ml falcon tubes containing food substrate; we adopted the same procedure for the 129 control individuals. We assessed the experiment in a thermostatic chamber at 25 ± 1°C, with a 130 14:10 hour light-dark cycle and with a humidity of 70%. After six days, we removed the adult 131 individuals and awaited the emergence of the newborns. We counted and noted newly emerged 132 adults daily. We performed five replicates for each condition. 133 134 2.4. Irradiation effect on D. melanogaster male’ longevity 135 To evaluate the effect of irradiation on the survival of D. melanogaster males, we compared the 136 average lifespan between irradiated and non-irradiated D. melanogaster males. We selected 72-96-137 hour-old D. melanogaster males that were irradiated as described in the previous paragraph at the 138 following doses: 80 Gy, 100 Gy, 120 Gy, 140 Gy, 160 Gy, 180 Gy. For each dose, we set up an 139 experimental cage (30 x 30 x 30 cm) with 20 individuals each. Two other cages were set up as 140 controls, in which we placed non-irradiated individuals: one cage was called “home control” with 141 individuals maintained at constant conditions of the thermostatic chamber, and one cage called “trip 142 control”, with individuals that we transported to the ENEA Calliope facility, but outside of the 143 irradiation unit. The “trip control” allowed us to evaluate if the transport could induce an impact on 144 the longevity of the individuals. For all conditions, we carried out mortality checks every day until 145 all the individuals died. 146 147 2.5. Irradiation effect on D. melanogaster male’ courtship behavior 148 We conducted courtship experiments to evaluate the time spent courting D. suzukii females by D. 149 suzukii and irradiated D. melanogaster males and to assess potential differences in the courtship 150 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 7 time in relation to the administered irradiation doses. We selected individuals following the 151

of the previous analyses. In this experiment, we irradiated 72-96 hours old D. 152 melanogaster males only at 80 Gy, 160 Gy and 180 Gy. We did not consider irradiations at 100, 153 120, and 140 Gy because they did not lead to significant differences in terms of sterility and 154 longevity (see Results section). 155 We set up “no-choice” and “choice” experimental trials: in the “no-choice” condition, we 156 placed one D. suzukii female with homospecific or heterospecific male into a falcon (15 mL) and 157 analyzed the male courtship time. Specifically, we analyzed: - the courtship time of D. suzukii male 158 with a D. suzukii female; - the courtship time of D. melanogaster male irradiated at 80, 160 and 180 159 Gy with a D. suzukii female. In the “choice” conditions, we placed one D. suzukii female with two 160 homospecific or heterospecific males into a falcon (15 mL) to evaluate the male's courtship time. 161 Specifically, we analyzed: - the courtship time of two no-irradiated D. suzukii males with a D. 162 suzukii female; - the courtship time of one no-irradiated D. suzukii male and one D. melanogaster 163 male irradiated at 80, 160 and 180 Gy with a D. suzukii female. For the observation of behaviors 164 between two D. suzukii males, due to their morphological similarity, the videos were analyzed at 165 reduced playback speed to track the individuals and annotate their behaviors accurately. 166 Conversely, in the second condition involving heterospecific males, the two species were 167 distinguishable: D. suzukii males possess characteristic black spots on their wings (hence the name 168 "spotted-wing drosophila"), which are absent in D. melanogaster males. For all conditions, 169 following a 5-minute acclimation period, we recorded the individual's behavior for 10 minutes 170 using an Olympus Tough TG-6 camera. After recording, we analyzed the videos using the Boris 171 software (Behavioral Observation Research Interactive Software), taking into account the courtship 172 elements such as orientation, touch, wing scissoring, wing spreading, and copulation attempt 173 (35,36). We carried out 20 replications for each trial to ensure the robustness of our data. 174 175 2.6. Data analysis 176 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 8 In the sterility experiment, the sterility degree achieved by D. melanogaster males was 177 evaluated for each experimental cond ition, applying a GLM model (generalized linear model with 178 negative binomial distribution), and the model family was selected comparing the AIC and BIC 179 estimators and the likelihood ratio test. We performed Tukey's multiple comparison test as a post 180 hoc test using the ‘multcomp’ package (37). The average percentage of residual fertility in each 181 condition was obtained by calculating the percentage reduction of each replicate in a specific 182 condition compared to the mean of offspring born in the control condition (i.e. 100% fertility) and 183 calculating the mean (± SE) of the percentages obtained in each replicate. 184 To evaluate the effect of the irradiation dose on the longevity of D. melanogaster males, 185 survival distributions of the different D. melanogaster groups (‘Control cages’, ‘Control trip’, ‘80 186 Gy’, ‘100 Gy’, ‘120 Gy’, ‘140 Gy’, ‘160 Gy’, ‘180 Gy’) were computed using the Kaplan-Meier 187

with the ‘ survival’ package and the differences between survival distributions were 188 estimated using the Log-Rank Test with the ‘survminer’ package (38,39). 189 In the courtship experiment, to compare the courtship time of D. suzukii and D. melanogaster 190 males in “no-choice” condition, we used a GLM model (generalized linear model with negative 191 binomial distribution), selecting the model family based on the AIC and BIC estimators and the 192 likelihood ratio test. We performed Tukey's multiple comparison test as a post hoc test. In the 193 “choice” condition, we used a GLM model (generalized linear model with negative binomial 194 distribution) to compare the average courtship time of males. Then, we compared the average 195 courtship time of the two males in the same condition using the nonparametric statistical Wilcoxon 196 Signed Rank test using the ‘dplyr’ package (40). All analyses were carried out using R Software 197 version 3.6.2. (41). 198 199 3. Results 200 3.1. Drosophila melanogaster males’ sterilization 201 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 9 We assessed the sterility of D. melanogaster males by mating them with D. melanogaster females 202 at irradiation doses of 80, 100, 120, 160, and 180 Gy, with a non-irradiated control group for 203 comparison. The mean number of the emerged adults in the control condition was 171.8 (± 24.47) 204 (mean ± SE) (Table 1). A significant emergence reduction was observed at all irradiation doses. At 205 the 80 Gy irradiation condition, the mean number of emerged adults was 29.2 (± 9.25), while at 206 180 Gy irradiation, it dropped to 0.8 (± 0.58) (Table 1; Fig 1). The GLM model showed a 207 significant effect of the male irradiation dose on the number of offspring produced by D. 208 melanogaster females (Table 2). Tukey's multiple comparison test showed a significant offspring 209 reduction from the control condition to all irradiation conditions, i.e. 80 Gy (z = 4.253, p = <0.001), 210 100 Gy (z = 5.183, p = <0.001), 120 Gy (z = 5.634, p = <0.001), 140 Gy (z = 6.660, p = <0.001), 211 160 Gy (z = 7.194, p = <0.001) and 180 Gy (z = 8.317, p = <0.001). There were also significant 212 differences between the 80 Gy irradiation dose and 160 Gy (z = 3.497, p= 0.008) and 180 Gy (z = 213 5.535, p= <0.001) doses. The 180 Gy dose showed significant differences with 80 Gy (see above), 214 100 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 doses, but not with 160 Gy. We did not observe significant differences between 80, 100, 120 and 216 140 Gy (Fig 1). 217 218 Table 1 . The mean number of the emerged D. melanogaster 219 adults. Mean number (±SE) of emerged adults and average 220 percentage (±SE) of the residual fertility at the different treatment 221 doses (Gy). 222 Treatment Dose (Gy) Mean number of emerged adults (±SE) Average percentage (±SE) of residual fertility 0 Gy 171.8 (± 24.47) 100 % 80 Gy 29.2 (± 9.25) 17 % (± 5.38) .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 10 100 Gy 23.5 (± 5.52) 11.29 % (± 3.45) 120 Gy 19.5 (± 2.25) 9.19 % (± 2.38) 140 Gy 11.75 (± 2.66) 5.59 % (± 1.73) 160 Gy 5.25 (± 2.39) 3.05 % (± 24.47) 180 Gy 0.8 (± 0.58) 0.47 % (± 0.34) 223 224 Table 2. Irradiation effect on D. melanogaster male’ sterility. GLM model values are shown. 225 Values in boldface indicate significant differences. 226 Fixed Effects Estimate ±SE z Value p-Value (Intercept) 2.9653 0.3051 9,718 < 2e-16 120 Gy -0.2053 0.4342 -0.473 0.63643 140 Gy -0.7035 0.4435 -1.586 0.11272 160 Gy -1.3070 0.4942 -2.645 0.00817 180 Gy -3.1884 0.6526 -4.886 1..03e-06 80 Gy 0.4089 0.4275 0.956 0.33882 No irradiation 2.1811 0.4208 5.183 2.18e-07 227 228 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 229 Fig 1. Irradiation effect on D. melanogaster male’ sterility. The number of D. melanogaster adults 230 emerged from fertile females and irradiated males. Different letters mean significant differences by Tukey 231 Multiple Comparison tests (p < 0.05). 232 233 3.2. Irradiation effects on D. melanogaster male’s longevity 234 In the longevity tests of D. melanogaster males, Kaplan- Meier curves showed significant 235 differences in lifespan between the treatments (80 Gy, 100 Gy, 120 Gy, 140 Gy, 160 Gy, 180 Gy, 236 the ‘home control and ‘trip control’ conditions) (Mantel-Cox log-rank; χ 2 = 105.9, d.f.= 7, P = < 2e-237 16) (Fig 2). The pairwise comparison test showed that control individuals have a higher probability 238 of survival than irradiated individuals. In particular, the ‘trip control’ condition showed significant 239 differences with all the irradiation doses tested (80 Gy, p = 0.0 39; 100 Gy, p = 0.027; 120 Gy, p = 240 0.013; 140 Gy, p = 0.017; 160 Gy, p < 0.001; 180 Gy, p < 0.001; ‘home control’ condition, p = 241 0.014), with an average life of 71 days. Instead, the ‘home control’ condition showed significant 242 differences only with 160 Gy (p = 0.001), 180 Gy (p = 0.027), and the ‘trip control’ condition, with 243 an average life of 68 days. Significant differences were also observed in the longevity of males 244 irradiated at 80 Gy and those irradiated at 160 Gy (p < 0.001), 180 Gy (p < 0.001), and the ‘trip 245 lts ey nt y, - ity nt = = nt ith es ip .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint control’ condition. The longevity of individuals irradiated at 160 Gy is significantly different from 246 both control treatments and 80 Gy irradiation and also from 100 Gy (p < 0.001), 120 Gy ( p < 247 0.001), 140 Gy (p < 0.001) and 180 Gy (p = 0.013). Furthermore, we found that the longevity of 248 individuals irradiated at 180 Gy is significantly different from both control treatments, 80 Gy and 249 160 Gy, and also from 100 Gy (p < 0.001), 120 Gy (p < 0.001), and 140 Gy (p < 0.001 ). The 250 average life of individuals irradiated at 160 Gy was 46 days and irradiated at 180 Gy was 55 days. 251 We found no significant differences between the dose irradiations 80, 100, 120 and 140 Gy, with an 252 average life of 63 days (Fig 2). 253 254 Fig 2. Irradiation effect on D. melanogaster male longevity. Kaplan-Meier curves showing the effect of 255 different irradiation doses on the longevity of D. melanogaster males. 256 257 3.3. Irradiation effect on D. melanogaster male’ courtship behavior 258 In the “no-choice” conditions, the mean (± SE) courtship time widely ranged from 16.70% (± 3.98) 259 (D. melanogaster irradiated at 160 Gy) up to 67.66% (± 8.30) (D. suzukii males) (Fig 3). The GLM 260 model showed significant differences in the average courtship time among conditions (Table 3). 261 Tukey's multiple comparison tests showed a significant difference between D. suzukii homospecific 262 m < of nd he s. an of 8) M ). fic .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 13 condition and the conditions with D. melanogaster irradiated at 160 Gy (z = 4.346, p = <0.001) and 263 180 Gy (z = 3.552, p = 0.002) and also a significant difference between D. melanogaster irradiated 264 at 80 Gy and 160 Gy (z = 2.697, p = 0.035). No significant differences were observed in terms of 265 courtship time between D. suzukii males and D. melanogaster males irradiated at 80 Gy (p > 0.05) 266 and between the courtship time of D. melanogaster irradiated at 80 and 180 Gy (p > 0.05) (Fig 3). 267 In the “choice” trials, the mean courtship times varied across treatments. In the conspecific 268 condition with two D. suzukii males, the mean courtship time was 24.23% (± 6.17) for one male and 269 16% (± 3.50) for the other. In the heterospecific condition with irradiated D. melanogaster males at 270 80 Gy, the D. suzukii male exhibited a mean courtship time of 12.60% (± 3.06), while the D. 271 melanogaster male displayed 31.83% (± 7.37). In the heterospecific condition with irradiated D. 272 melanogaster males at 160 Gy, the D. suzukii male showed a mean courtship time of 24% (± 4.88), 273 whereas the D. melanogaster male had 9.66% (± 4.61). In the heterospecific condition with D. 274 melanogaster males irradiated at 180 Gy, the courtship time was 12.59% (± 1.94) for the D. suzukii 275 male and 13.58% (± 5.68) for the D. melanogaster male (Fig 4). 276 The GLM analysis revealed significant differences in the average courtship time among conditions 277 (Table 4), and Tukey’s multiple comparisons test indicated a significant difference only between the 278 courtship times of irradiated D. melanogaster males irradiated at 80 Gy and 160 Gy (p = 0.016; Fig 279 4). The Wilcoxon rank sum test highlighted significant differences only in the total courtship time 280 between D. melanogaster males irradiated at 160 Gy and D. suzukii males (W = 82.5, p = 0.002) 281 (Fig 4). 282 283 Table 3. Irradiation effect on D. melanogaster male’ courtship time in “no-choice” 284 condition. GLM model values are shown. Values in boldface indicate significant differences. D. 285 suz = Drosophila suzukii; D. mel = Drosophila melanogaster. 286 Fixed Effects Estimate ±SE z Value p-Value .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 14 (Intercept) 2.8151 0.2273 12,384 < 2e-16 D. mel 180 Gy 0.2592 0.3204 0.809 0.41853 D. mel 80 Gy 0.8595 0.3188 2.697 0.00069 D. suz (homospecific condition) 1.3994 0.3220 4.346 1.39e-05 287 288 Table 4. Irradiation effect on D. melanogaster male’ courtship time in “choice” condition. 289 GLM model values are shown. Values in boldface indicate significant differences. D. suz = 290 Drosophila suzukii; D. mel = Drosophila melanogaster. 291 Fixed Effects Estimate ±SE z Value p-Value (Intercept) 3.4604 0.2447 14,139 < 2e-16 D. suz 80 Gy -0.9271 0.3496 -2.652 0.00799 D. mel 160 Gy -1.1924 0.3513 -3.394 0.00068 D. suz 160 Gy -0.2828 0.3469 -0.815 0.41493 D. mel 180 Gy -0.8515 0.3538 -2.407 0.01609 D. suz 180 Gy -0.9279 0.3496 -2.654 0.00794 D. suz 1 (homospecific condition) -0.6934 0.3530 -1.964 0.04948 D. suz 2 (homospecific condition) -0.2730 0.3514 -0.777 0.43724 292 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 293 Fig 3. Courtship comparison in the “no-choice” trials. Courtship of D. suzukii males toward D. suzukii294 females (pink column); courtship of D. melanogaster males toward D. suzukii females at different irradiation 295 doses (blue, green and orange columns). *** Tukey's multiple com- parison tests p < 0.001; ** Tukey's 296 multiple comparison tests p < 0.01; * Tukey's multiple comparison tests p-value < 0.05. Black dots are box -297 plot outliers. 298 299 kii on y's - .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 300 Fig 4. Courtship comparisons in “choice” trials. Time spent courting D. suzukii females by D. suzu kii301 males and irradiated D. melanogaster males at 80, 160 and 180 Gy. D. suz = D. suzukii males (pink 302 columns); D. mel 80 Gy = D. melanogaster males irradiated at 80 grey (blue column); D. mel 160 Gy = D. 303 melanogaster males irradiated at 160 grey (green column); D. mel 180 Gy = D. melanogaster males 304 irradiated at 180 grey (orange column). Black dots are box-plot outliers. * Tukey's multiple comparison 305 tests p-value < 0.05; ** Wilcoxon rank sum test p < 0.01. 306 307 4. Discussion 308 Finding the best irradiation dose is a crucial issue that requires careful evaluation to develop a 309 heterospecific SIT approach. We found that irradiation was highly effective at reducing fertility. All 310 irradiation doses led to a significant reduction in adult emergence with respect to the control 311 condition (Table 1; Fig 1). We observed at the lower irradiation dose tested (80 Gy), only a 17% (± 312 5.38) average residual fertility that decreases as the irradiation doses increase until reaching 0.47 (± 313 0.34) average residual fertility at the highest dose tested (180 Gy) (Table 1). These results are 314 consistent with previous findings. Studies about the effect of gamma rays on the sterility of D. 315 kii nk D. es on a ll ol (± (± re D. .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 17 melanogaster have been carried out in the 1960s and ‘70s. Henneberry (1963) (42) found that D. 316 melanogaster irradiated males at 160 Gy dose produced nonviable eggs after mating with non-317 irradiated females. Accordingly, Nelson (1973) (43) observed that at 120 Gy 99.3% fewer progeny 318 emerged than non-irradiated individuals. 319 A trade-off between the sterility and longevity of the irradiated males is critical in 320 optimizing classic and heterospecific SIT applications (44). The survival analysis showed that 321 irradiation significantly reduces the lifespan of D. melanogaster males, with the highest reductions 322 in longevity at the highest doses. Control individuals lived on average for 70 days, whereas males 323 irradiated at the highest doses (160–180 Gy) experienced a 50-day lifespan (Fig 2). This 324 observation aligns with prior studies indicating that irradiation-induced oxidative stress and cellular 325 damage can impair physiological functions, shortening lifespan (45). Nelson et al. (1973) (43) also 326 reported decreased longevity in irradiated D. melanogaster, with a similar reduction in lifespan at 327 the highest dose tested of 150 Gy. The dose-dependent decrease in longevity must be carefully 328 considered when applying SIT since male competitiveness may be compromised if they do not 329 survive long enough to mate effectively in the wild. A reduction in the average lifespan of D. 330 melanogaster from 70 in the controls to 50 days at the highest radiation doses can be seen unlike to 331 compromise the effectiveness of SIT, as frequent releases of sterile individuals are typically part of 332 the strategy. For instance, regarding screwworm Cochliomyia hominivorax (Coquerel), the releases 333 have to occur weekly to maintain the critical ratio or even twice a week for the Mediterranean fruit 334 fly and tsetse Glossina austeni (Wiedemann) (46–48). We want to highlight that this study was not 335 addressed to assess the longevity of sterile individuals in field conditions, which can be lower than 336 in protected field-cage situations, where sterile males have easy access to food and are protected 337 from predation in the laboratory (49). This aspect certainly warrants further investigation. 338 The last part of this study was designed to assess the heterospecific courtship behavior of 339 irradiated D. melanogaster males. A balance between sterility and behavioral competence when 340 selecting an irradiation dose for pest control is critical. If males lose the ability to court females, 341 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 18 their sterility will have limited impact on population suppression, as seen in studies of other insect 342 species, such as the Queensland fruit fly ( Bactrocera tryoni Froggatt), where high doses impaired 343 sexual competitiveness (50). Our findings showed that irradiated D. melanogaster males retained 344 their ability to court D. suzukii females even at the highest irradiation doses, suggesting that 345 courtship behavior remains largely unaffected by irradiation. In the “no-choice condition”, however, 346 D. melanogaster males irradiated at 160 and 180 Gy exhibited significantly lower courtship activity 347 compared to D. suzukii males toward conspecific females (Table 3; Fig 3). Conversely, under the 348 “choice condition”, D. melanogaster males courted D. suzukii females as much as D. suzukii males, 349 even at the highest irradiation doses tested. Notably, D. melanogaster males irradiated at 160 Gy 350 showed reduced courtship toward D. suzukii females compared to D. melanogaster males irradiated 351 at 80 Gy and D. suzukii males, corroborating the observations made in the “no-choice condition” 352 (Fig 4). These results suggest two key points. First, the presence of D. melanogaster males seems to 353 influence the courtship behavior of D. suzukii males, as they courted conspecific females more in 354 the “no-choice condition” compared to the “choice condition”. The reduced courtship behavior 355 observed at a radiation dose of 160 Gy suggests that higher doses may lead to behavioral 356 impairments in D. melanogaster males. These impairments are likely attributable to physiological 357 alterations or disruptions in neural circuits essential for mating displays. Ionizing radiation is known 358 to damage neural pathways involved in courtship behavior, as evidenced in moths, where higher 359 doses often result in physiological defects that reduce their competitiveness with wild populations 360 (51,52). Radiation may also interfere with producing or expressing key biochemical and behavioral 361 signals. During courtship, D. melanogaster males emit specific biochemical signals, such as cis-362 vaccenyl acetate, along with behavioral signals like wing vibrations and pheromone release, to 363 stimulate female responses (53–55). Ionizing radiation may disrupt these signals, compromising the 364 male's ability to communicate with females effectively. Similar disruptions have been observed in 365 other pest species, such as Callosobruchus chinensis L. females and Anthonomus grandis 366 (Boheman) males, where radiation-induced impairments in mating signals led to reduced courtship 367 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 19 and mating success (56,57). Consequently, irradiated D. melanogaster males may fail to elicit 368 appropriate responses from females, disrupting courtship and mating dynamics. In our study, while 369 D. melanogaster males irradiated at 160 Gy showed reduced courtship, males exposed to 180 Gy 370 courted D. suzukii females comparably to untreated D. suzukii males under both "no-choice" and 371 "choice" conditions (Figs 3 and 4). Additionally, the highest courtship percentage was observed at 372 the lowest tested dose of 80 Gy (Figs 3 and 4), supporting the notion that lower radiation doses may 373 preserve male courtship behavior more effectively. 374 Overall, our study highlights the complex interactions between irradiation, longevity, 375 sterility, and mating behavior in D. melanogaster and contributes to growing evidence of using 376 heterospecific SIT in pest control. Based on our results, the 80 Gy and 180 Gy radiation doses 377 appear most suitable for further investigation. At 80 Gy, we observed the highest courtship rates 378 and longest lifespan among the radiation doses tested. In comparison, at 180 Gy, we achieved the 379 greatest reduction in fertility. 380 For an effective SIT program, it is essential that sterile males survive for a long time in their 381 environment. Only in this way they can mate with a sufficient number of wild females and induce 382 sterility in the population. If their quality is compromised and their longevity reduced, more 383 frequent and larger-scale releases will be required to sustain a high overflooding ratio, ultimately 384 increasing operational costs (58). Studies on An. arabiensis and Ae. aegypti have highlighted 385 different factors influencing longevity. An. arabiensis was found to have a significantly shorter 386 lifespan in field settings compared to laboratory conditions, whereas Ae. aegypti exhibited a 387 stronger sensitivity to seasonality (59). Specifically, in a field experiment carried out in Vietnam, 388 Ae. aegypti populations demonstrated significantly higher survival rates during the cool or hot dry 389 seasons compared to the cool and wet seasons (60). Moreover, a synergistic effect of irradiation, 390 packing, and chilling was observed to compromise the longevity of An. arabiensis —an effect that 391 was not detected in Ae. aegypti (59). These results suggest that the impact of irradiation on lifespan 392 is highly species-dependent. In our case, it is essential first to assess the differences in D. 393 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 20 melanogaster males' longevity kept under laboratory conditions and those exposed to environmental 394 conditions. This would allow us to better evaluate the lifespan reduction at 180 Gy and determine 395 whether it constitutes a limitation for SIT applications. Conversely, a higher residual fertility at 80 396 Gy than 180 Gy might be less restrictive given that we are not dealing with a pest or invasive 397 species. Unlike in standard SIT applications for pest control, achieving the high sterility levels 398 required to release pest males (as discussed by Bakri et al. 2021 (61) and Parker et al. 2021 (62)) 399 may not be necessary in this context. Based on these considerations, the present study has 400 demonstrated that an irradiation dose of 80 Gy seems to be more effective. However, further studies 401 are needed better to evaluate both longevity and mating performance under field conditions. 402 Greenhouses and other enclosed environments appear ideal for implementing the 403 heterospecific Sterile Insect Technique (h-SIT) to manage D. suzukii populations. Studies on 404 plastic- and mesh-covered tunnels have shown that mechanical barriers alone can significantly 405 reduce D. suzukii populations in these confined areas. This reduction is due not only to the physical 406 exclusion provided by the barriers but also to creating an unfavorable microclimate for the pest's 407 survival (63). Although complete exclusion cannot be achieved through mechanical barriers alone, 408 integrating h-SIT with these measures could enhance the overall effectiveness of biocontrol 409 strategies. 410 411

412 Our findings highlight the critical balance between sterility, longevity, and mating behavior in D. 413 melanogaster for heterospecific SIT applications. Among the tested doses, 80 Gy emerged as the 414 most effective, preserving male longevity and mating performance while significantly reducing 415 fertility. While 180 Gy achieved the highest sterility, the potential lifespan and courtship behavior 416 trade-offs warrant further evaluation. Future studies should focus on-field performance to refine SIT 417 protocols. Integrating h-SIT with mechanical barriers in controlled environments like greenhouses 418 .CC-BY 4.0 International licenseavailable under a (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 The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639671doi: bioRxiv preprint 21 could enhance D. suzukii management, making 80 Gy a promising dose for practical 419 implementation. 420 421 Acknowledgments 422 We thank Alessandra Spanò, Elisa Michelangeli and Giulia Pezzi for their technical help. 423 424 425 426 427

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