Production and shipment of Wolbachia-infected eggs allow controlling Aedes albopictus through the Incompatible Insect Technique on a remote island

Production and shipment of Wolbachia-infected eggs allow controlling Aedes albopictus through the Incompatible Insect Technique on a remote island | 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 Article Production and shipment of Wolbachia-infected eggs allow controlling Aedes albopictus through the Incompatible Insect Technique on a remote island Julien Cattel, Benjamin Gaudillat, Marianne Duployer, Sarah Scussel, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6936662/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Climate and land-use changes are accelerating the spread of the mosquito Aedes albopictus , a major arbovirus vector, leading to the emergence and autochthonous transmission of Dengue or Chikungunya viruses in temperate regions such as Italy and France. This situation is stimulating the development of innovative vector control strategies allowing to overcome the rapid selection of insecticide resistance. The Incompatible Insect Technique (IIT) allows suppressing mosquito populations through inundative releases of artificially Wolbachia infected males that sterilize local females through Cytoplasmic Incompatibility (CI). We carried out a six-month IIT suppression trial on a remote island located in the Western Indian Ocean. We used a recently constructed and optimized Aedes albopictus transinfected line sheltering a single Wolbachia infection and inducing bi-directional CI. This feature ensures that released males sterilize local females, while infected females resulting from accidental releases are also sterilized by wild-type males, thereby preventing population replacement, a key limitation of conventional IIT. The trial was conducted in operational conditions: mosquito populations were monitored during suppression and the number of released males was adjusted based on wild population density. Importantly, eggs were produced in a central insectary located over 1,000 km from the release area, transported via commercial flights to a satellite insectary for male production, and finally shipped by boat to the release site. Our results demonstrated that (i) over 95% suppression can be achieved within a few weeks of treatment, (ii) as expected the use of a mono-infected line prevented population replacement, (iii) large-scale shipment of eggs under operational conditions is both feasible and effective, supporting the scalability and industrial deployment of this environmental-friendly vector control strategy. Biological sciences/Biotechnology/Environmental biotechnology Biological sciences/Microbiology/Applied microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Aedes albopictus has for a long time been considered as a poor disease vector because of a zoophilic behaviour and a limited competence for the transmission of some arboviruses 1 . This mosquito species is being increasingly scrutinized because it has been the major vector of a number of dengue or chikungunya epidemics in tropical regions within the last decades. Most importantly, the plasticity and invasive behaviour of this vector together with accelerating global changes are leading to a rapid expansion of its geographic distribution, including in temperate countries where Ae. albopictus has proven efficient for autochthonous transmission of Dengue virus 2 , 3 . In the absence of available vaccines, transmission is routinely reduced by controlling vector populations using insecticides. However, more environmental-friendly methods, such as the Sterile Insect Technique (SIT) or the Incompatible Insect Technique (IIT) are being developed to bypass the selection of insecticide resistance and reduce the impacts of pesticides on non-target species (Dobson, 2021). Both methods rely on the release of sterile males to reduce the population size of the targeted species, offering the advantage of species-specific control allowing sterilization of females in cryptic habitats or effective control of insecticide-resistant mosquito populations 4 . IIT leverages the properties of Wolbachia pipientis , a maternally inherited endosymbiotic bacterium that infects approximately 40% of arthropod species 5 , 6 . Wolbachia can manipulate mosquito reproduction through Cytoplasmic Incompatibility (CI), a form of conditional embryonic lethality that occurs when a Wolbachia infected male mates with a female that is either uninfected or infected by a different, incompatible Wolbachia strain 5 . CI can be understood as resulting from a modification/rescue mechanism ( mod / resc ), wherein the mod function modifies the sperm during spermatogenesis and the resc function, expressed in the egg cytoplasm laid by Wolbachia -infected females, rescues the embryo through an interaction with the modified sperm 7 , 8 . CI can be hijacked for the development of the IIT, which involves the release of Wolbachia - infected males that are incompatible with resident females 9 , 10 . Recent studies have demonstrated the effectiveness of standalone IIT in controlling Ae . aegypti populations in the U.S 11 , Australia 12 , Singapore 13 , and Puerto Rico 14 as well as Ae . albopictus populations in China 15 . Importantly, all Wolbachia -transinfected lines used in the aforementioned field assays exhibit unidirectional CI with the resident females, meaning that accidentally released females are compatible with resident males, thus increasing the risk of population replacement with Wolbachia transinfected mosquitoes. To mitigate this risk, which may lead to IIT failure, it has been proposed to combine incompatible and sterile insect techniques, utilizing Wolbachia infection to induce male sterility and a low irradiation dose to sterilize those females that the sexing processes fail to eliminate 16 – 18 . An alternative approach is the exploitation of a bidirectional pattern of CI (Bi-CI), occuring when the native Wolbachia infection is removed and replaced by a foreign Wolbachia inducing CI 19 , 20 . In this case, transinfected females are only compatible with co-released transinfected males, significantly reducing the invasive potential of Wolbachia 21 . Indeed, assuming a similar fitness of natural and Wolbachia transinfected mosquitoes, Wolbachia infection frequency must exceed 50% to allow invasion 22 , compared to > 0% in the case of uni-CI. Under these circumstances, population replacement could occur only if the objective of IIT deployment is complete elimination, which to our knowledge has never been achieved in any SIT/IIT mosquito program 4 . When Bi-CI occurs, models suggest that immigration rates of approximately 2% of wild type individuals in the release area would be sufficient to mitigate the risk of population replacement in the long term 23 . Thus, the use of standalone IIT with a Wolbachia strain inducing Bi-CI is expected to achieve high levels of population suppression with limited risk of population replacement. However, this approach has been discussed primarily in theoretical terms 24 , 25 , and no field trials have yet demonstrated sustained, high-level suppression using stable bi-directional CI over the long term. In this context, we implemented a six-month IIT program aiming at controlling an Aedes albopictus population on a 9-hectare island in the Seychelles, utilizing a mono-transinfected mosquito line that induces bi-directional CI with the local population. We demonstrate that IIT deployment achieves population suppression reaching 95% within three months of the initial release, and a suppression level maintained above 50% until the end of the release period. Moreover, we show that while achieving a high suppression level over several months, the contamination percentage of incompatible individuals in the field never exceeds 7% and drops to 0% two months after the end of the releases. Finally, we demonstrate the feasibility of a mass production model in which mosquito eggs are produced at a central facility and subsequently distributed to a satellite rearing center dedicated to producing incompatible males only 26 , 27 . Altogether these results support the viability of this mass production model for industrial deployment. Results CI penetrance of the SEY- w Pip line Incompatible male releases were conducted using an Ae . albopictus w Pip transinfected line (Scussel et al., 2024) introgressed with a wild genetic background from the Seychelles, resulting in the SEY- w Pip line. Reciprocal crosses between wild lines and SEY- w Pip confirmed that w Pip induced bidirectional CI in Ae . albopictus regardless of the host genetic background 19 , 28 . Indeed, when SEY- w Pip males were crossed with wild females, they induced nearly complete CI, with a hatching rate ranging from 0.37 to 1.05%. Symmetrically, when wild males were crossed with SEY- w Pip females, the hatching rate ranged from 0.47 to 9,41% (Table 1 ). Table 1 Hatch rate obtained in control and reciprocal crosses between the SEY- w Pip and three different wild lines. crosses nb of hatched eggs nb of unhatched eggs hatch rate (%) (± SD) CI (%) CIcorr (%) ♂ SEY- w Pip x ♀ Le Port 78 9,053 1,05 (± 1,67) 98,95 92,93 ♂ SEY- w Pip x ♀ S-RUN 45 8,007 0,59 (± 0,70) 99,41 97,38 ♂ SEY- w Pip x ♀ La Providence 17 4,683 0,37 (± 0,07) 99,63 98,33 ♂ Le Port x ♀ SEY- w Pip 61 7,491 0,76 (± 0,31) 99,24 96,34 ♂ S-RUN x ♀ SEY- w Pip 25 5,401 0,47 (± 0,21) 99,53 97,74 ♂ La Providence x ♀ SEY- w Pip 465 4,562 9,41 (± 2,96) 90,59 54,69 ♂ Le Port x ♀ Le Port 10,468 1,701 85,14 (± 7,30) ♂ S-RUN x ♀ S-RUN 5,416 1,652 77,51 (± 4,92) ♂ La Providence x ♀ La providence 1,918 563 77,88 (± 5,80) ♂ SEY- w Pip x ♀ SEY- w Pip 9,875 2,483 79,23 (± 7,94) Life history traits (LHTs) and mating competitiveness of the SEY- w Pip line in laboratory conditions To assess the fitness of the SEY- w Pip line following introgression, we compared different LHTs with those of two wild lines, S-RUN and Le Port. The traits analyzed included longevity, number of laid eggs and hatching rate. For both sexes, Le Port line exhibited greater longevity than the S-RUN and SEY- w Pip lines (log-rank test; P < 0.001 for both comparisons), and SEY- w Pip also had a longer longevity than S-RUN, although this difference was significant in males only (Supp. Figure 1 A-B). We also observed a significant difference in the number of laid eggs between Le Port line, recording an average of 47.25 (± 14.65) eggs laid per female, and both SEY- w Pip and S-RUN lines, for which similar number of eggs were laid, 40.52 (± 12.30) and 42.19 (± 16.01), respectively (GLMM; quasi-poisson family; P = 0.627) (Supp. Figure 1 C). We observed a similar hatch rates in Le Port and S-RUN lines, with a mean of 83.97% (± 19.25) and 82.94% (± 21.05), respectively. These hatch rates were significantly higher than those observed for the SEY- w Pip line showing a mean value of 75.89% (± 25.75) (Supp. Figure 1 D). Finally, we assessed the mating competitiveness of SEY- w Pip males compared to wild-type males from the S-RUN and Le Port lines, by crossing virgin wild females with increasing ratios of incompatible (SEY- w Pip) to compatible (S-RUN or Le Port) males. Observed hatch rates at 1 : 0 and 0 : 1 ratios (where the first and second numbers correspond to the proportions of compatible and incompatible males, respectively) were compared with the expected hatch rates under the assumption that both male types had equal mating competitiveness. As expected, hatch rates decreased with increasing proportions of incompatible males, in crosses involving wild females from S-RUN or Le Port lines (Supp. Figure 1 E). Of note, when different combinations of SEY- w Pip and S-RUN males were used, observed hatch rates were consistently and significantly lower than expected, indicating that incompatible males were more competitive than S-RUN males. In crosses involving SEY- w Pip and Le Port males, observed and expected hatch rates were not different at 1:5 and 1:10 ratios. However, at a 1:1 ratio, the observed hatch rate was 20% higher than expected. Altogether, SEY- w Pip appears as a mono-transinfected line suitable for mass-production and subsequent use in an IIT program, as the potential fitness cost associated with w Pip infection and/or genetic introgression are absent or minimal. Estimation of the natural population size before releases The density of mosquito population at the release site was evaluated using mark-release-recapture (MRR) experiments. A total of 4,389 and 5,682 marked males were released during the first and second MRR experiment, conducted in October and November 2022, respectively performed 9 and 8 months before the first release. For both MRRs, the recapture rate of marked males was 1.5% over four consecutive days (68/4389 for the first and 87/5682 for the second MRR). Based on the first MRR, we estimated a total number of wild males on the whole island of 9,994 (± 1,984), while the second yielded an estimate of 15,540 (± 3743). We used the data of the second MRR to calibrate the number of incompatible males to be released to ensure a minimum incompatible male ratio of 5:1. This number was estimated to ~ 80,000 males per week (15,540*5). Shipping of eggs, release of incompatible males and suppression of the Ae . albopictus population The control site consists of a 7-hectare area in the Southern part of Saint Anne island, while the release site encompassed the entire 9-hectare area of Moyenne island. These sites are separated by 1.2 kilometers (Fig. 1 ). The mosquito population dynamics in both sites was monitored weekly before male releases, using ovitraps from 15th February, 2022 to 27th June, 2023. During this period, population trends were similar (Supp. Figure 2 A), as supported by the absence of significant difference in the number of hatched eggs between the two sites (Wilcoxon rank sum test; P = 0.608), and confirming the suitability of the selected control site. Likewise, no significant difference was observed in egg hatching rates between the release (95.27 ± 5.09%) and control (94.87 ± 5.79%) sites during the prerelease phase (GLMM-quasi-binomial family; P = 0.947) (Supp. Figure 2 B). Three months before the start of incompatible male releases, eggs of the SEY- w Pip line, continuously produced in Saint-Denis (La Réunion) were shipped weekly to the Seychelles in bubble wraps via commercial airlines. Over nine months of shipping, a total of approximately 145g of eggs (~ 4 g per week) was transported. Delivery time ranged from 7 to 15 days, depending on the regulatory inspection process at the Seychelles airport. Eggs were sent out in 32 shipments with an overall sending success rate of 90% (29/32), meaning that the observed hatching rate was close to the expected value. Releases of incompatible males started on 13th July 2023, and were maintained for six consecutive months. The release period was eventually separated in three phases, with distinct numbers of released males (Fig. 2 A). Approximately 80,000 males were released every week for twelve weeks during phase I. We observed an immediate suppression in the first week following the first release, with a 30% decrease in the hatch rate at the release site. By the second week, the hatch rate had dropped by 60%. At that time, we measured a male release ratio of 23:1 (SEY- w Pip to wild males). Then the hatch rate continued to decline throughout phase I, reaching 18.65% at the end of phase I (three months after the first release), compared to 93.10% in the control site (Fig. 2 B). Although the number of hatched eggs temporarily decreased in the control site during the first few weeks after the first release, it remained significantly lower in the release site throughout phase I (13th July- 29th September, 2023) ( P < 0.001) (Fig. 2 C). The impact of IIT deployment became particularly obvious from the eighth week following the first release, even if the male release ratio dropped to 1.5:1 (SEY- w Pip to wild males). At this point, the average number of hatched eggs in the control site began to increase weekly, reaching a mean of 49.15 (± 33.92) by the end of phase I. In contrast, the number of hatched eggs remained consistently low in the release site, with a mean of 3.08 (± 8.27) during the same period. Additionally, at the end of the phase I, 12 out of 26 ovitraps in the release site contained no hatched eggs. At this point the male release ratio increased to 7:1 (SEY- w Pip to wild males). After twelve weeks of releases, this resulted in an overall population suppression level of 95% (Fig. 2 C). After achieving a population suppression of 95%, we reduced the number of incompatible males released to approximately 50,000 per week, for six consecutive weeks, corresponding to phase II (Fig. 2 A). During this phase, we were able to maintain control over the population size, as evidenced by a significant difference in the number of hatch eggs between the release and the control sites (Wilcoxon rank sum test; P < 0.001). However, towards the end of phase II, we observed an increase in the number of hatched eggs in the release site during the final three weeks, coinciding with a lower male release ratio of 1:1 (SEY- w Pip to wild males). Between the end of phase I and phase II the hatch rate increased by 23%, while the suppression level decreased by 35% (Fig. 2 B-C), with a male release ratio remaining at 1:1 (SEY- w Pip to wild males) by the end of phase II. In response to the decline in effectiveness, we increased the number of incompatible males released to approximately 140,000 per week for the last four weeks of releases, corresponding to phase III. This adjustment significantly restored control efficiency, stabilizing the hatch rate and increasing the population suppression level, which reached 80% by the end of releases. During phase III, we also observed a significant difference in the number of hatched eggs between the release and control sites (Wilcoxon rank sum test; P < 0.001). To investigate the duration of suppression following the end of releases, we continued to monitor the population for ten additional weeks. During this post-release survey, we observed a significant difference in the number of hatched eggs between sites (Wilcoxon rank sum test; P < 0.001). However, starting from the seventh week after the last release, the hatch rate and the number of hatched eggs in the release site became comparable to those observed in the control site. Spatial heterogeneity of mosquito suppression An analysis of spatial population dynamics revealed that egg hatch was impacted by the release of incompatible males at all surveyed ovitraps. However, we found that this impact was heterogeneous, with some areas being more difficult to maintain at low densities throughout the entire release period, particularly in the southwestern part of the release site (Fig. 3 ). Absence of population replacement The experimental set-up allowed addressing whether standalone IIT using a transinfected line exhibiting bi-CI could strongly suppress a field population without causing population replacement. To assess this, we firstly quantified weekly the female contamination rate in the male pupae production following mechanical sex separation. The female contamination rate ranged from 0.015 to 0.08% throughout the entire mass production period (Fig. 4 A). During this quality control of the production, all pupae identified as females were removed. However, since not all male pupae were verified and because human verification is inherently imperfect, the release of accidental females did occur as shown by the presence of w Pip females at the study site (see below). Thus, to monitor the risk of SEY- w Pip mosquito establishment in the field, we collected and reared all larvae from eggs found in ovitraps at the release site every three weeks, from phase I through the end of the post-release phase. Larvae were reared until adulthood to determine the percentage of w Pip-positive individuals per ovitrap. In total, this monitoring was conducted seven times during the releases and four times during the post-release period, representing 1,196 screened adults. We observed a w Pip-positive rate ranging from 0 to 6.3% during the release period, with a maximum of three positive ovitraps (Fig. 4 B). Although w Pip-positive individuals were detected several weeks after the last release, their frequency never exceeded 7%. Importantly, no positive samples (= 176) were found in the final screening conducted more than two months after the last release. Moreover, analysis of the spatial distribution of positive ovitraps revealed that they were located in different areas across screening, with only one ovitrap testing positive on two consecutive occasions (Supp. Figure 3 ). These findings support the hypothesis of an absence of local establishment and instead suggest sporadic releases, involving one or a few females released at different locations from week to week. Discussion Aedes albopictus is playing a major role in the emergence or re-emergence of dengue and chikungunya in tropical regions and has also been implicated in outbreaks in newly-colonized temperate regions such as Italy in 2007 29–32 . Although proof-of-concept examples remain limited, the Incompatible Insect Technique (IIT) and the Sterile Insect Technique (SIT) have already demonstrated their effectiveness in controlling Ae . albopictus populations (Aldridge, Gibson, & Linthicum, 2024). One of the main advantages of IIT over SIT is that males are “ready to use”. In SIT programs, the rearing facility producing sterilized insects must be located near the release sites, or sterile males must be transported over long distances, in which case handling conditions (such as chilling and compaction) need to be optimized and transport minimized to prevent adverse effects on males 33 . However, SIT offers the sustained advantage over IIT that accidentally released female are sterile. This limitation of IIT is particularly salient in the case of uni-directional CI, which to our knowledge has been thus far used in all long-term published IIT studies aiming at suppressing Ae . albopictus populations. To overcome this limitation, we implemented a long term standalone IIT program for the control of Ae . albopictus using a transinfected line exhibiting bi-CI with the local population. We conducted this trial in “real-life” conditions, meaning through the monitoring of mosquito dynamics during suppression and subsequent adjustment of release conditions. The release of SEY- w Pip males led to a 95% population suppression within only twelve weeks. This level of suppression was reached although the estimated male release ratio was low, 1.5:1 and 7:1 (SEY- w Pip to wild males), 8 and 12 weeks after the first release, respectively. These results suggest that the mono-transinfected line used in this study allowed efficient suppression even when using low incompatible to wild male ratios. Such a performance is in keeping with previously published data showing that the introgression of a genetic background from the targeted population does limit fitness costs associated with Wolbachia transinfection 28 , 34 – 36 . After achieving a substantial level of population suppression (phase I), we choose to decrease the number of released males from ~ 80,000 to 50,000 per week (phase II). Although at this time the number of wild mosquitoes was low due to efficient suppression, the number of hatching eggs rapidly increased as exemplified by two consecutive measures following releases with low SEY- w Pip to wild males ratio (1:1), potentially due to favorable climatic conditions offered by the onset of the rainy season. Finally, we increased the number of incompatible males to ~ 140,000 per week (phase III) for the last three releases, allowing restoring 80% suppression. Altogether, these data indicate that, despite a significant reduction of the wild population size, a minimal incompatible male ratio should be conserved for a long term population control. Interestingly, we showed that the suppression effect was heterogeneous at the release site, potentially resulting from habitat heterogeneity, as reported in other IIT program 15 . We observed that ovitrap M01, located in the southwest part of the release site, displayed consistently lower suppression than surrounding ovitraps. We hypothesized that this difference resulted from favorable breeding conditions around this trap, neighboring a bamboo area (where cut bamboo stems provide larval sites unless they are refilled). This interesting pattern suggests that constant monitoring of the treated areas should be associated with local tuning of the number of released males, with increasing numbers being used in areas identified as resisting to mosquito suppression. While presented data show the effectiveness of a standalone IIT for the control of an Ae . albopictus population in an insular context, we demonstrate that a significant population suppression can be achieved without population replacement. Indeed, although we detected w Pip-positive individuals during and shortly after the release period (ranging from 0 to 6.3%), no incompatible individuals were found in our final screening conducted two months after the last release. We acknowledge that visual inspection of a large number of pupae performed in this study is time consuming and can be hardly implemented at substantially larger scales. To overcome this limitation, it has been proposed to implement an individual automated verification at the adult stage allowing to reduce drastically the female contamination rate 11 , 14 , 37 . Classically, female pupae contamination after classic mechanical sex separation ranges from 0.5 to 3% 11,38 . Here we demonstrated that female contamination can be reduced to 0.02%, collecting pupae on two consecutive days for each production batch. Lastly, this program was carried out on a remote island, representing one of the most challenging scenarios for the deployment of IIT as population replacement cannot be mitigated by the migration of wild mosquitoes surrounding the treated area. Presented data highlight another advantage of the deployment of a standalone IIT: the possibility of developing a mass production model based on a unique central facility for eggs production associated with satellite units dedicated to the production of incompatible males. Such satellite units are relatively easy to set-up as there is little need for space and labor as equipments for blood-feeding or egg collection are not unnecessary 27 . As compared to the shipping of chilled adults from a central facility to satellite units, the shipping of eggs, facilitated by the resistance of Ae . albopictus eggs to desiccation and their low weight, ensures the production of high quality males while avoiding a quality control on males post-shipping. This model of mass production can also be developed in SIT programs under the strict condition that an irradiator is present at each satellite unit, which is clearly challenging but should be considered in the case of a mosquito extinction program. Methods Mosquito lines We firstly constructed a transinfected line with a wild genetic background from the Seychelles. For this, we used a laboratory line transinfected with w Pip-IV 19 , a Wolbachia strain naturally found in Culex pipiens 39 . Females from this line were backcrossed with males from a wild line sampled using ovitraps on Ile Longue (Seychelles), located about 400 meters from the release site. Backcrossing was performed using 15 day-old wild males (using the F 1 post sampling) with 4–5 day-old virgin females from the transinfected line for four consecutive generations, resulting in a line named SEY- w Pip, expected to display 93,75% of the wild nuclear genetic background. We used three other wild lines, named S-RUN, Le Port, and La Providence, to conduct laboratory experiments aimed at quantifying CI level induced by the SEY- w Pip line and at measuring possible fitness costs following introgression. The S-Run and Le Port lines were originally derived from a natural population on Reunion Island 28 , 40 , and were maintained for 50 and 10 generations, respectively, before experimentation. The La Providence line was sampled from the Providence neighborhood on Mahé island (the largest island in the Seychelles archipelago) and maintained for 7 generations before experiments. Adults of all lines were maintained at a temperature of 27 ± 1◦C, a relative humidity of 70 ± 5% and a 12 : 12 h light:dark photoperiod in the insectary located at Saint-Denis (Reunion island). Females were blood-fed using the Hemotek system (Hemotek Ltd, United Kingdom), bovine blood provided by the regional slaughterhouse (Saint-Pierre, Reunion island) and supplemented with EDTA (0,1%). CI expression of the transinfected line To quantify CI penetrance of the SEY- w Pip line following introgression, we performed reciprocal en masse crosses using S-RUN, Le Port and La Providence lines. Although en masse crosses can mask individual variability, especially in the case of moderate CI, complete or nearly complete CI were expected 19 , which led us to favor en masse rather than individual crosses in order to screen a larger number of mosquitoes. Crosses involved 2-5-day-old virgin females and males (100 from each sex) in 15 × 15 × 15 cm cages (Bugdorm, Taiwan). Three replicates were performed for each cross. Females were given a blood meal 48h after mating and eggs were collected 5 days later. After 7 days of drying, eggs were allowed to hatch for 24 h (in a jar containing 250 mL tap water supplemented with 50 mg of TetraMin (TETRA) and the number of hatched and unhatched eggs was counted. A hatch rate was calculated as follows: hatch rate = (number of hatched eggs/total number of eggs) × 100. To account for the embryonic mortality not related to CI, we used a corrected CI index (CI corr ) 41 , 42 calculated as follows: CI corr = [(CI obs − CCM)/(100 − CCM)] × 100, where CI obs is the percentage of unhatched eggs observed in a given incompatible cross, and CCM is the mean mortality observed in the control crosses. Life history traits of the transinfected line Longevity and fecundity of the SEY- w Pip line were compared to those of the S-RUN and Le Port lines, both showing high fitness under laboratory-controlled conditions 19 , 28 . Larvae-rearing conditions were standardized between lines for all experiments. Specifically, eggs from each line were allowed to hatch for 24 h at 31°C in containers containing 250 ml of water supplemented with 50 mg TetraMin (TETRA). For each line, 2,500 L1 were manually counted and transferred to a tray (53 × 325 × 65 cm, MORI 2A) and fed with a controlled quantity of food (TetraMin (TETRA), day 1: 0.45 g, day 2: 1 g, day 3: 1.25 g, day 4: 1 g, day 5: 1 g, day 6: 0.75 g, day 7: 0.5 g) until pupal stage. Male and female pupae were initially separated using a pupae sex sorter (Wolbaki, WBK-P0001-V1 model), and then individually inspected under a binocular loupe. For each line, longevity was measured by introducing 100 newly emerged males or females separately in 15 × 15 × 15 cm cages (Bugdorm, Taiwan) in which sugar meal (5%) was changed weekly. Three replicates were performed at different times for each line and sex and longevity was determined by recording the number of dead mosquitoes for 45 days. For each line, the number of laid eggs was measured by placing 200 male and 200 female pupae (one cage per line) in 30 × 30 × 30 cm cages (Bugdorm, Taiwan) and left for 3 days following emergence. A blood meal was then provided and engorged females were placed in a separate cage. Five days later, 50 females were randomly selected and placed individually in a small plastic cup for egg laying. Cups with at least one laid egg were conserved for the analyses. After 7 days of drying, eggs were counted, allowed to hatch for 24 h and hatch rates were measured. Three replicates were performed at different times for each line. Mating competitiveness of incompatible males in laboratory-controlled conditions We evaluated the mating competitiveness of incompatible males by mixing virgin females from the S-RUN or Le Port line with increasing ratios of males from the SEY- w Pip line. Pupae from both lines were allowed to emerge in separate 30 × 30 × 30 cm cages (Bugdorm, Taiwan) and adults were provided sugar meal (5%). Mating competitiveness was monitored using 2-3-day old virgin females and males. A hundred males were first placed inside cages followed by the release of an equal number of females, and mating was allowed for 48 h. All males were then removed, a blood meal was provided to females and eggs were collected by oviposition en masse 5 days later. After 7 days of drying, eggs were allowed to hatch for 24 h and the hatching rate was measured and used as a proxy of mating competitiveness. Five ratios of males were tested: 1 : 1 (50♂ S-RUN or Le Port : 50♂ SEY- w Pip), 1 : 5 (17♂ S-RUN or Le Port : 85♂ SEY- w Pip), 1 : 10 (9♂ S-RUN or Le Port: 90♂ SEY- w Pip) and two control ratios, 1 : 0 (100♂ S-RUN or Le Port: 0♂ SEY- w Pip) and 0 : 1 (0♂ S-RUN or Le Port: 100 SEY- w Pip). Three replicates in different times were performed for each cross and ratio. Description of study areas Both study sites were located within the Sainte Anne Marine National Park in the Seychelles. The control site consists of a 7-hectare area in the Southern part of Saint Anne island, while the release site encompassed the entire 9-hectare area of Moyenne island (Fig. 1 ). These sites, separated by 1.2 kilometers, presented comparable ecological conditions, with similar vegetation and geology, providing suitable climatic conditions for Ae . albopictus populations. The region is classified as having a tropical maritime climate, with year-round temperatures ranging from 22°C to 33°C. The rainy season extends from November to April, peaking in January and February, while the dry season lasts from May to October. Relative humidity is generally high, often exceeding 80%. Experiment approvals were provided by the Seychelles Bureau of Standards (SBS) and the National Biosecurity Agency (NBA), in accordance with local regulations on insect population release and monitoring. Estimating Ae . albopictus natural population size at the release site The natural population size of Ae . albopictus at the release site was estimated through two mark-release-recapture (MRR) experiments conducted prior releases. Adults used for MRR were obtained from eggs collected in the wild via ovitraps, which were deployed to monitor the temporal dynamics of Ae . albopictus population size at the release site (see below). Larvae were reared under laboratory-controlled conditions until pupal stage. Male and female pupae were initially separated using a pupae sex sorter (Wolbaki, WBK-P0001-V1 model), and all male pupae were individually inspected under a binocular loupe. To mark adult males with fluorescent pigment, we constructed a self-marking unit adapted from a previously described prototype 43 . A total of 2,500 male pupae was placed in a square petri dish (12.5 x 12.5 cm) with approximately 0.5 cm water, which was previously positioned inside a 30 x 30 x 30 cm cage (Bugdorm-1 model) (Bugdorm, Taiwan). A removable exit grid, consisting of wool yarns lines spaced approximately 0.5 cm apart and pre-coated with fluorescent pigment (Radiant color), was then placed above the petri dish. Upon emergence, adult males were self-marked as they passed through the grid and were subsequently conserved in the cage with a sugar solution 5% until release. The self-marking unit was then removed from the cage, and the number of males that had not crossed the grid was counted. Just before the release, dead adults were removed from the cage to ensure an accurate count of the alive marked males being released. Altogether, 4,389 and 5,682 marked males were released during the first and second MRR experiments, conducted in October and November 2023, respectively. For both MRRs, males were released at four equidistant spots, surrounded by 27 BG traps (Supp. Figure 4 ), corresponding to an area of ~ 4.5 ha. Just after releases, each BG trap was activated for four days of capture, with a collection bag being replaced every 24 h. All captured individuals were frozen at -20°C for 24h, species and sex of each trapped adult was determined under a binocular loupe. All Ae . albopictus males were then examined under ultraviolet light to detect the presence of fluorescent dust. The population size was estimated as follows: N = n * ( M / m ), where N is the population size per day, n the number of captured mosquitos, M the number of released marked mosquitos, and m the number of recaptured marked mosquitos 44 – 46 . Mass Production and release of incompatible males Mass production and subsequent release of SEY- w Pip males involved five steps: (i) egg production at the CYROI insectary (Saint-Denis, Reunion island), (ii) transportation of eggs to Seychelles using a commercial airline company, (iii) larvae rearing and sex separation at insectary of the Ministry of Health of Seychelles (Grand Anse, Seychelles), (iv) transportation of male pupae by boat to the release site, and (v) release of adult males. For egg production, we maintained adult cages (45×45×45 cm) (Bugdorm, Taiwan) continuously, each containing approximately 6,000 female pupae and 2,000 male pupae. Adults were fed with a 5% sugar solution. Females were blood-fed three times a week with bovine blood, and eggs were collected weekly for three consecutive weeks. Eggs were matured for one week before being used either for the production of adults used for egg production or sent to Seychelles for the production of incompatible males. The procedures for egg hatching, larvae rearing and sex separation were similar in both distant insectaries. Briefly, the eggs were brushed and transferred in 120 ml plastic containers with a hatching solution (tap water with TetraMin (TETRA) at 1g/L) and left for 8 h. Approximately 3,000 larvae were added to each tray (53 × 325 × 65 cm, MORI 2A) and fed with a controlled quantity of food (TetraMin (TETRA), day 1: 0.5 g, day 2: 1 g, day 3: 1.25 g, day 4: 1.5 g, day 5: 0.75 g, day 6: 1 g, day 7: 0.75 g, day 8: 0.5 g) until pupal stage. Male and female pupae were separated using a pupae sex sorter (Wolbaki, WBK-P0001-V1 model). During the release phase, after the initial mechanical sex separation, quality control was performed daily on all (or nearly all, depending on the production phase; see below) collected pupae considered as male pupae, by measuring female contamination under a binocular loupe. Using a volumetric template, daily male pupae production was divided into several lots of approximately 2,250 male pupae, placed into 500 mL containers (Nalgene) with ~ 2 cm water depth. The following day, all pupae were transported by boat to the release site, and each container was transferred into a release tube (10-cm diameter x 35-cm height). The tubes were placed in trays containing ~ 2 cm water depth, and a source of 10% sugar solution was placed at the top. The following day, the release tube was removed from the water and adult males were released. All male pupae produced weekly were released over two consecutive days, with the release evenly distributed across the 40 release spots, spaced approximately 40 meters apart (Supp. Figure 5). Incompatible males were released over a period of six months, divided into three phases, each distinguished by the number of released males. Phase I involved the release of approximately 80,000 males/week over 12 weeks. Phase II involved the release of around 50,000 males/week over 6 weeks. Finally, phase III involved the release of approximately 140,000 males/week over the last four weeks (Fig. 2 A). For phases I and II, quality control of the male pupae production after mechanical sex separation was performed on the entire production, while in phase III, quality control was conducted on only two-thirds of the production. Measure of the incompatible vs. wild male ratios during the release period The incompatible vs. wild male ratios were measured monthly throughout the entire release period (i.e. five measurements), from July 2023 to November 2023. For this, 9 BG-Sentinel traps (Biogents, Regensburg, Germany) were distributed across the release site, spaced approximately 90 meters apart, resulting in one trap per hectare (Supp. Figure 6). BG traps were activated shortly after each release and for 24-h of capture. Captured mosquitoes were then morphologically identified under a binocular loupe to determine sex and species of each specimen. All Ae . albopictus adult males were individually placed in 1.5 ml Eppendorf tubes and stored at -20°C before being shipped to Reunion island for Wolbachia typing through PCR analysis as previously described 47 . Monitoring population suppression Temporal dynamics of the Ae. albopictus population size in both the control and release sites were monitored using ovitraps before, during, and after the release periods. Ovitraps consisted of small black 800-mL buckets containing 500 mL of tap water and one egg-laying paper (Sartorius™) fixed with a piece of plexiglas. From February 2022 to February 2024, 26 and 20 ovitraps were distributed throughout the release and control sites, respectively, with ovitraps placed approximately 50 meters apart, resulting in approximately 3 ovitraps per hectare (Supp. Figure 7A-B). Ovitraps were retrieved and replaced weekly. Positive egg-laying papers, defined as those containing at least one egg, were preserved, and the number of eggs was recorded. Eggs were then stored at room temperature for six days, and then allowed to hatch for 24 h in a jar containing 250 mL tap water supplemented with 50 mg of TetraMin (TETRA). Hatch rate was then calculated as described previously 28 . To measure the suppression efficiency, we calculated the suppression percentage using the following equation, where Hc and Hr represent the average number of hatched eggs per trap at the control and release sites, respectively 15 : $$\:SE\left(\text{%}\right)=\frac{Hc-Hr}{Hc}x100$$ Monitoring the risk of population replacement The risk of population replacement at the release site, caused by accidental releases of transinfected females, was monitored from the beginning until two months after the last release (from 2 August 2023 to 20 February 2024). During this period, every three weeks, all larvae from the 26 ovitraps were conserved and reared until adult stage. Adults were then frozen for 1 h at -20°C, individually transferred to 1.5 mL Eppendorf tube and shipped to Reunion island laboratory facilities for PCR screening. The dynamics of w Pip-positive adults were monitored at eleven different time points, with a total of 1,196 individuals checked for the presence or absence of w Pip. Two indicators were used to monitor the risk: (i) the proportion of ovitraps containing w Pip-positive adults and (ii) the proportion of w Pip-positive individuals emerging from each positive ovitrap. DNA extraction and w Pip screening Genomic DNA was extracted from adult mosquitoes using the cetyltrimethylammonium bromure (CTAB) method 48 . The PCR reaction mixture consisted of 2 µl gDNA template, 8.5 µl nuclease free water, 1 µl of each primer (10 µM), and 12.5 µl of GoTaq® G2 Hot Start Master Mixes (Promega corporation). We targeted an ankirin domain protein by amplifying the ank2 gene using the following primers: F-CTTCTTCTGTGAGTGTACGT and R2-TCCATATCGATCTACTGCGT 49 . PCR amplification was performed under the following conditions: 95°C 5 min followed by 35 cycles of 94°C 45 s, 53°C 45 s, 72°C 45 s, and 72°C for 7 min. Amplified fragments were run in agarose gel (1.5%) electrophoresis. Statistical analysis Longevity data were analysed using a log-rank test. Fecundity data (count data) were analysed using a generalized linear mixed model (GLMM, quasi-poisson family, log link) in which the different mosquito lines were included as a fixed effect and the repetitions (individual egg laying) as a random factor. We used a GLMM to analyse egg hatch rate (binary data, quasi-binomial family, logit link). The overdispersion of the data was checked using a R code proposed by Ben Bolker and others ( https://bbolker.github.io/mixedmodels-misc/glmmFAQ.html ). A F-test for the GLMM-quasi-binomial and quasi-poisson models were used to analyse deviance. Exact binomial tests were used to compare the observed and expected hatch rates in the mating competitiveness experiment (proportional data). Analyses were performed in R version 4.3.1 50 , using lme4 package for all mixed models 51 , MASS package 52 for using the quasi-binomial and quasi-poisson families in the GLMMs, and survival package for longevity data analyses 53 . For all data, the significance level was set to α = 0.05. Declarations Acknowledgements We gratefully acknowledge the Seychelles Bureau of Standards (SBS), the Ministry for Agriculture, Climate Change and Environment (MACCE), and the National Biosecurity Agency (NBA) for granting the necessary permits in Seychelles. We also extend our sincere thanks to the Seychelles Parks and Gardens Authority (SPGA), Club Med Sainte Anne, and the Moyenne Island Foundation for facilitating access to the Sainte Anne Marine National Park. We thank the Ministry of Health (MoH) and the University of Seychelles (UniSey) for their support throughout the project. This project was financially supported by the European Union, La Région Réunion, and Le Département de La Réunion. It was funded through two European Regional Development Fund (ERDF) Interreg programs: the SeyWol Project – Phase I (INTERREG V OCEAN INDIEN 2014–2020, No. 0031783) and the Operating SeyWol Project (INTERREG VI OCEAN INDIEN 2021–2027, No. 004954), as well as by the Fonds de Coopération Régionale 2021 (No. 001749). Finally, we warmly thank all individuals who contributed to the project, including Mr. David Labrosse from the Ministry of Health, and the staff at Jolly Roger Bar & Restaurant for their kind support. Author contributions J.C. contributed to the study concept and design, conducted laboratory experiments and mass mosquito production, analyzed and interpreted the data, and wrote and prepared the manuscript and figures. B.G. contributed to the study design, analyzed and interpreted the data and was responsible for mass production of incompatible males and entomological surveys in Seychelles. M.D., D.S., A.J-B., and S.D. contributed to the mass production and release of incompatible males, as well as to entomological monitoring in Seychelles. M.A., H.D., H.P., K.S. and S.S. contributed to the entomological surveys. L.B. assisted in obtaining the necessary approvals to implement the field trial in Seychelles. J.F. and G.R. contributed to the logistical and technical administrative aspects. S.S., Q.L., and J.E. contributed to laboratory experiments, mass egg production in La Réunion, and molecular biology analyses. P.M. contributed to the initiation of the project in Seychelles. P.T. also contributed to the project’s initiation and to the preparation of the manuscript. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at References Paupy, C., Delatte, H., Bagny, L., Corbel, V. & Fontenille, D. Aedes albopictus , an arbovirus vector: From the darkness to the light. Microbes Infect. 11 , 1177–1185 (2009). Rezza, G. Aedes albopictus and the reemergence of Dengue. BMC Public Health 12 , 72 (2012). Kraemer, M. U. G. et al. Past and future spread of the arbovirus vectors Aedes aegypti and Aedes albopictus . Nat. Microbiol. 4 , 854–863 (2019). Aldridge, R. L., Gibson, S. & Linthicum, K. J. Aedes Aegypti controls Ae . Aegypti : SIT and IIT-an overview. J. Am. Mosq. Control Assoc. 40 , 32–49 (2024). Werren, J. H. Biology of Wolbachia . Annu. Rev. Entomol. 124 , 587–609 (1997). Zug, R. & Hammerstein, P. Still a host of hosts for Wolbachia : Analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PLoS One 7 , 7–9 (2012). Beckmann, J. F., Ronau, J. A. & Hochstrasser, M. A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility. Nat. Microbiol. 2 , 17007 (2017). LePage, D. P. et al. Prophage WO genes recapitulate and enhance Wolbachia -induced cytoplasmic incompatibility. Nature 543 , 243–247 (2017). Boller, E. F., Russ, K., Vallo, V. & Bush, G. L. Incompatible races of European cherry fruit-fly, Rhagoletis cerasi (Diptera-Tephritidae), their origin and potential use in biological-Control. Entomol. Exp. Appl. 20 , 237–247 (1976). Riegler, M. & Stauffer, C. Wolbachia infections and superinfections in cytoplasmically incompatible populations of the European cherry fruit fly Rhagoletis cerasi (Diptera, Tephritidae). Mol. Ecol. 11 , 2425–2434 (2002). Crawford, J. E. et al. Efficient production of male Wolbachia -infected Aedes aegypti mosquitoes enables large-scale suppression of wild populations. Nat. Biotechnol. 38 , 482–492 (2020). Beebe, N. W. et al. Releasing incompatible males drives strong suppression across populations of wild and Wolbachia -carrying Aedes aegypti in Australia. Proc. Natl. Acad. Sci. U. S. A. 118 , e2106828118 (2021). Consortium, P. W.--S. & Ching, N. L. Wolbachia -mediated sterility suppresses Aedes aegypti populations in the urban tropics. Medrxiv 2006–2021 (2021). Ryff, K. R. et al. Incompatible Aedes aegypti male releases as an intervention to reduce mosquito population — A field trial in Puerto Rico. 19 , e 00128391 (2025) doi:10.1371/journal.pntd.0012839. Zeng, Q. et al. A standalone incompatible insect technique enables mosquito suppression in the urban subtropics. Commun. Biol. 5 , 1–13 (2022). Zheng, X. et al. Incompatible and sterile insect techniques combined eliminate mosquitoes. Nature 572 , 56–61 (2019). Bansal, S. et al. Effectiveness of Wolbachia -mediated sterility coupled with sterile insect technique to suppress adult Aedes aegypti populations in Singapore: a synthetic control study. Lancet Planet. Heal. 8 , e617–e628 (2024). Chansang, C. et al. Combined sterile insect technique and incompatible insect technique: The first proof-of-concept to suppress Aedes aegypti vector populations in semi-rural settings in Thailand. PLoS Negl. Trop. Dis. 13 , 1–21 (2019). Scussel, S. et al. Combining transinfected Wolbachia and a genetic sexing strain to control Aedes albopictus in laboratory-controlled conditions. Proc. R. Soc. B 291 , 20240429 (2024). Calvitti, M., Moretti, R., Lampazzi, E., Bellini, R. & Dobson, S. L. Characterization of a new Aedes albopictus (Diptera: Culicidae)– Wolbachia pipientis (Rickettsiales: Rickettsiaceae) symbiotic association generated by artificial transfer of the w Pip strain from Culex pipiens (Diptera: Culicida. J. Med. Entomol. 47 , 179–187 (2010). Dobson, S. L., Fox, C. W. & Jiggins, F. M. The effect of Wolbachia -induced cytoplasmic incompatibility on host population size in natural and manipulated systems. Proc. R. Soc. B Biol. Sci. 269 , 437–445 (2002). Hoffmann, A. A. & Michael, T. Cytoplasmic incompatibility in insects. Influ. passengers 42–80 (1997). Moretti, R., Marzo, G. A., Lampazzi, E. & Calvitti, M. Cytoplasmic incompatibility management to support Incompatible Insect Technique against Aedes albopictus . Parasites and Vectors 11 , (2018). Li, Y. et al. Reply to: Issues with combining incompatible and sterile insect techniques. Nature 590 , E3-E5 (2021). Moretti, R. & Calvitti, M. Issues with combining incompatible and sterile insect techniques. Nature 590 , E1–E2 (2021). Denton, J. A., Joubert, D. A., Goundar, A. A. & Gilles, J. R. L. International shipments of Wolbachia infected mosquito eggs: towards the scaling-up of World Mosquito Program operations. OIE Rev. Sci. Tech. 41 , 91–99 (2022). Dobson, K. L. et al. Satellite rearing of Aedes mosquito eggs: synchronized empirical test of a novel mass rearing model. J. Am. Mosq. Control Assoc. 39 , 12–17 (2023). Lejarre, Q. et al. Development of the Incompatible Insect Technique targeting Aedes albopictus : introgression of a wild nuclear background restores the performance of males artificially infected with Wolbachia . Appl. Environ. Microbiol. e02350-24 (2025). Goubert, C., Minard, G., Vieira, C. & Boulesteix, M. Population genetics of the Asian tiger mosquito Aedes albopictus , an invasive vector of human diseases. Heredity (Edinb). 117 , 125–134 (2016). Rezza, G. et al. Infection with chikungunya virus in Italy: an outbreak in a temperate region. Lancet 370 , 1840–1846 (2007). Frumence, E. et al. Genomic insights into the re-emergence of chikungunya virus on Réunion Island, France, 2024 to 2025. Eurosurveillance 30 , 1–6 (2025). Santé publique France (SpF). Bilan de la surveillance renforcée de la dengue , du chikungunya et du Zika en France hexagonale du 1 er mai au 17 juin 2025 Chikungunya , dengue et Zika Episodes de transmission autochtone. 1–8 (2025). French. Available from: https://www.santepubliquefrance.fr/maladies-et-traumatismes/maladies-a-transmission-vectorielle/chikungunya/documents/bulletin-national/chikungunya-dengue-et-zika-en-france-hexagonale.-bulletin-de-la-surveillance-renforcee-du-17-juin-2025 Guo, J., Zheng, X., Zhang, D. & Wu, Y. Current Status of Mosquito Handling, Transporting and Releasing in Frame of the Sterile Insect Technique. Insects 13 , 532 (2022). Benedict, M. Q. Sterile Insect Technique: Lessons from the Past. J. Med. Entomol. 58 , 1974–1979 (2021). Garcia, G. de A. et al. Matching the genetics of released and local Aedes aegypti populations is critical to assure Wolbachia invasion. PLoS Negl. Trop. Dis. 13 , 1–23 (2019). Ross, P. A. Designing effective Wolbachia release programs for mosquito and arbovirus control. Acta Trop. 222 , 106045 (2021). Beebe, N. W. et al. Releasing incompatible males drives strong suppression across populations of wild and Wolbachia -carrying Aedes aegypti in Australia. Proc. Natl. Acad. Sci. U. S. A. 118 , (2021). Gong, J.-T. et al. Upscaling the production of sterile male mosquitoes with an automated pupa sex sorter. Sci. Robot. 9 , eadj6261 (2024). Atyame, C. M., Delsuc, F., Pasteur, N., Weill, M. & Duron, O. Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito. Mol. Biol. Evol. 28 , 2761–2772 (2011). Lebon, C., Benlali, A., Atyame, C., Mavingui, P. & Tortosa, P. Construction of a genetic sexing strain for Aedes albopictus : A promising tool for the development of sterilizing insect control strategies targeting the tiger mosquito. Parasites and Vectors 11 , (2018). Poinsot, D., Bourtzis, K., Markakis, G., Savakis, C. & Merçot, H. Wolbachia T ransfer from Drosophila melanogaster into D . simulans : H ost E ffect and C ytoplasmic I ncompatibility R elationships. Genetics 150 , 227–237 (1998). Cattel, J. et al. Back and forth Wolbachia transfers reveal efficient strains to control spotted wing drosophila populations. J. Appl. Ecol. 55 , 2408–2418 (2018). Vavassori, L., Saddler, A. & Müller, P. Active dispersal of Aedes albopictus : A mark-release-recapture study using self-marking units. Parasites and Vectors 12 , 1–14 (2019). Seber, G. A. F. The estimation of animal abundance and related parameters . (1982). Pollock, K. H., Nichols, J. D., Brownie, C. & Hines, J. E. Statistical inference for capture-recapture experiments. Wildl. Monogr. 3–97 (1990). Cianci, D. et al. Estimating mosquito population size from mark-release-recapture data. J. Med. Entomol. 50 , 533–542 (2013). Atyame, C. M. et al. Comparison of irradiation and Wolbachia based approaches for sterile-male strategies targeting Aedes albopictus . PLoS One 11 , 1–15 (2016). Collins, F. et al. Construction of a general human chromosome jumping library, with application to cystic fibrosis. Science 235 , 1046–1050 (1987). Duron, O. et al. Variability and expression of ankyrin domain genes in Wolbachia variants infecting the mosquito Culex pipiens . J. Bacteriol. 189 , 4442–4448 (2007). R Core Team. R: A Language and Environment for Statistical Computing. (2023). Bates, D. et al. Package ‘lme4’. Convergence 12 , 2 (2015). Ripley, B. et al. Package ‘mass’. Cran r 538 , 113–120 (2013). Therneau, T. M. A Package for Survival Analysis in R. (2023). Additional Declarations There is NO Competing Interest. Supplementary Files dataLHTfecundityhatchratelabSuppFig1.csv Dataset 2 dataCIcrosses.xlsx Dataset 1 dataLHTmalecompetitivenesslabSuppFig1.csv Dataset 4 datawPipfemalecontaminationreleasesiteFig4.xlsx Dataset 7 datanumberhatchedeggshatchrateFig2.csv Dataset 6 dataMRR.xlsx Dataset 5 dataLHTlongevitylabSuppFig1.csv Dataset 3 SupplementaryFigures.docx Supplementary figures Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6936662","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":478562218,"identity":"a7857aeb-2ba1-426e-b6a4-b549619e96ee","order_by":0,"name":"Julien Cattel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIie2PsWrDMBCGTwiUxUnnTH6FeCw09qvUCDIl76BgkBelsz3lLUxHiYNOeQMt8RvIWxJSqJOSQqDIGQvVN/zcwX38HEAg8Achggp9m7Xrk40GFfKtRAzAVBeFPtADNwWjyzSk0Cpf6+49jbO40PhyauIJBeK6paejyoWpdzxRjL3i6s0mkgKd1o1fwbHURLFohitlSa8wOh5SPqXOFHty+Kxs9phCpM77FkA42HxYUa0wG8m5+ljMzEZYLikpvL8kJUd3lOm8LLDdH852vi0L4zqfIu465TXFb5c/xPfr2XscCAQC/5QvyG9XozXu7zYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8715-586X","institution":"SAS SYMBIOTIC","correspondingAuthor":true,"prefix":"","firstName":"Julien","middleName":"","lastName":"Cattel","suffix":""},{"id":478562219,"identity":"a138e8f1-c5d4-4377-ab83-bf27f1a66cc8","order_by":1,"name":"Benjamin Gaudillat","email":"","orcid":"","institution":"Groupement d’intérêt Public Cyclotron Océan Indien (CYROI)","correspondingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"","lastName":"Gaudillat","suffix":""},{"id":478562220,"identity":"420536a3-e789-4787-8f19-89fca1fc80ff","order_by":2,"name":"Marianne Duployer","email":"","orcid":"","institution":"Groupement d’intérêt Public Cyclotron Océan Indien (CYROI)","correspondingAuthor":false,"prefix":"","firstName":"Marianne","middleName":"","lastName":"Duployer","suffix":""},{"id":478562221,"identity":"77a35bf2-fc32-4678-a2d0-813f1924027f","order_by":3,"name":"Sarah Scussel","email":"","orcid":"","institution":"Groupement d’intérêt Public Cyclotron Océan Indien (CYROI)","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Scussel","suffix":""},{"id":478562222,"identity":"bf28828a-3c9f-423c-a0c6-14539fb4daf0","order_by":4,"name":"Quentin Lejarre","email":"","orcid":"","institution":"SAS SYMBIOTIC","correspondingAuthor":false,"prefix":"","firstName":"Quentin","middleName":"","lastName":"Lejarre","suffix":""},{"id":478562223,"identity":"262a196d-d249-439c-8a4b-160201963237","order_by":5,"name":"Jeremy Esnault","email":"","orcid":"","institution":"Groupement d’intérêt Public Cyclotron Océan Indien (CYROI)","correspondingAuthor":false,"prefix":"","firstName":"Jeremy","middleName":"","lastName":"Esnault","suffix":""},{"id":478562224,"identity":"1e2e7819-6046-422d-b99a-7428742051c1","order_by":6,"name":"Daryl Sinon","email":"","orcid":"","institution":"Island Biodiversity and Conservation center (IBC)","correspondingAuthor":false,"prefix":"","firstName":"Daryl","middleName":"","lastName":"Sinon","suffix":""},{"id":478562225,"identity":"2a72a7f9-10bb-435b-901d-e981511cbbdb","order_by":7,"name":"Alvin Jean-Bonnelame","email":"","orcid":"","institution":"Island Biodiversity and Conservation center (IBC)","correspondingAuthor":false,"prefix":"","firstName":"Alvin","middleName":"","lastName":"Jean-Bonnelame","suffix":""},{"id":478562226,"identity":"8b7d56b5-9b5a-4894-b94a-4fa7518bdae9","order_by":8,"name":"Stéphanie Donet","email":"","orcid":"","institution":"Island Biodiversity and Conservation center (IBC)","correspondingAuthor":false,"prefix":"","firstName":"Stéphanie","middleName":"","lastName":"Donet","suffix":""},{"id":478562227,"identity":"2163b684-a3cc-46c4-9b7f-32f36dfc1971","order_by":9,"name":"Merna Amade","email":"","orcid":"","institution":"Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Merna","middleName":"","lastName":"Amade","suffix":""},{"id":478562228,"identity":"313e5d2f-e06e-45e7-a197-8e71d209d0c7","order_by":10,"name":"Hilary Desir","email":"","orcid":"","institution":"Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Hilary","middleName":"","lastName":"Desir","suffix":""},{"id":478562229,"identity":"2a2df3c0-53bd-4ea0-af83-b44801dbd165","order_by":11,"name":"Hamid Pool","email":"","orcid":"","institution":"Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Hamid","middleName":"","lastName":"Pool","suffix":""},{"id":478562230,"identity":"667ddbdf-161e-4ed4-a00b-28a9f57e2b66","order_by":12,"name":"Kenneth Sinon","email":"","orcid":"","institution":"Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Kenneth","middleName":"","lastName":"Sinon","suffix":""},{"id":478562231,"identity":"4820425e-1833-4a43-9b90-ca5f8c0f7f7f","order_by":13,"name":"Steeve Savy","email":"","orcid":"","institution":"Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Steeve","middleName":"","lastName":"Savy","suffix":""},{"id":478562232,"identity":"74315590-8c47-4909-b461-eebc8267d8a8","order_by":14,"name":"Nigel Sultan","email":"","orcid":"","institution":"Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Nigel","middleName":"","lastName":"Sultan","suffix":""},{"id":478562233,"identity":"471d532b-76a9-41ab-acc3-723c71c6e230","order_by":15,"name":"Kérina Jean-Baptiste","email":"","orcid":"","institution":"Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Kérina","middleName":"","lastName":"Jean-Baptiste","suffix":""},{"id":478562234,"identity":"98506391-87aa-4961-9c49-7c10bd714ddf","order_by":16,"name":"Leon Biscornet","email":"","orcid":"https://orcid.org/0000-0001-6074-6582","institution":"Seychelles Public Health Laboratory, Public Health Authority, Ministry of Health","correspondingAuthor":false,"prefix":"","firstName":"Leon","middleName":"","lastName":"Biscornet","suffix":""},{"id":478562235,"identity":"562672a1-3dd7-494c-a5fc-b69bbc6ebcb4","order_by":17,"name":"Joseph François","email":"","orcid":"","institution":"Island Biodiversity and Conservation center (IBC)","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"François","suffix":""},{"id":478562236,"identity":"1611de55-79dd-433e-8b7f-e63c1b462dda","order_by":18,"name":"Gerard Rocamora","email":"","orcid":"","institution":"Island Biodiversity and Conservation Centre, University of Seychelles","correspondingAuthor":false,"prefix":"","firstName":"Gerard","middleName":"","lastName":"Rocamora","suffix":""},{"id":478562237,"identity":"5c098ce1-3449-404e-8f1b-1b791fce907c","order_by":19,"name":"Patrick MAVINGUI","email":"","orcid":"","institution":"UMR PIMIT (Processus Infectieux en Milieu Insulaire Tropical)","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"MAVINGUI","suffix":""},{"id":478562238,"identity":"ca736719-09ba-42e7-a11a-d8c0befd94db","order_by":20,"name":"Pablo Tortosa","email":"","orcid":"","institution":"Université de La Réunion, Unité Mixte de Recherche Processus Infectieux en Milieu Insulaire Tropical (UMR PIMIT), CNRS 9192, INSERM 1187, IRD 249","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Tortosa","suffix":""}],"badges":[],"createdAt":"2025-06-20 08:00:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6936662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6936662/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-09269-0","type":"published","date":"2025-11-26T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87207189,"identity":"dbdbba35-57c3-4cb8-9017-22031c314654","added_by":"auto","created_at":"2025-07-21 14:16:08","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":308917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eControl and release sites, located within the Sainte Anne Marine National Park in the Seychelles. \u003c/strong\u003eThe control site (7 ha) is a part of Sainte-Anne island, while the release site corresponds to the entire surface of Moyenne island.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/1d820206b7383cf06e55e8c2.jpeg"},{"id":87203805,"identity":"35345c85-4587-44bd-882c-e7c1a3232b2c","added_by":"auto","created_at":"2025-07-21 13:44:08","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":474678,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppression of the wild population.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Total number of SEY-\u003cem\u003ew\u003c/em\u003ePip males released each week during the release period. \u003cstrong\u003eB\u003c/strong\u003eAverage hatching rate per week in the release and control sites. \u003cstrong\u003eC\u003c/strong\u003e The average number of hatched eggs per week and suppression efficiency. The red dashed line represents the suppression efficiency on the release site compared to the control site. Vertical blue and green lines represent standard deviation.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/40d695fa654e2673bb0ab01d.jpeg"},{"id":87204387,"identity":"4eb77d9b-68ba-476f-8ac5-ab06cde7a966","added_by":"auto","created_at":"2025-07-21 13:52:08","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":136015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial-temporal dynamics of hatched eggs at the release and control sites for each release phase.\u003c/strong\u003e The different color indicates the average number of hatched eggs.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/d01e25b958010613f9fd561f.jpeg"},{"id":87204384,"identity":"79479e1a-38dc-474e-b1bb-0407f490076f","added_by":"auto","created_at":"2025-07-21 13:52:08","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":310714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemporal dynamics of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ew\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePip females in laboratory male production and at the release site. A \u003c/strong\u003eFemale contamination rates measured in SEY-\u003cem\u003ew\u003c/em\u003ePip males during the mass production. For each week of mass production, we indicated the total number of pupae individually inspected under a binocular loop and between brackets the percentage of pupae individually inspected by the total number of adult released males. \u003cstrong\u003eB \u003c/strong\u003e\u003cem\u003ew\u003c/em\u003ePip-positive individual rate detected at the release site measured using ovitraps. For each screening we indicated the number of \u003cem\u003ew\u003c/em\u003ePip-positive individuals and the total number of screened adults. Between brackets: total number of ovitraps for which at least one \u003cem\u003ew\u003c/em\u003ePip-positive individual was detected.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/da630979d43ec9a114230f83.jpeg"},{"id":99211852,"identity":"b99d6572-8c0b-4685-9c15-74e5835b4d3b","added_by":"auto","created_at":"2025-12-30 08:10:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2659808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/f1012a4b-8017-43a7-bfc1-9eafe6057333.pdf"},{"id":87203800,"identity":"365bfa17-7dbf-4355-9fdc-65960af7fc9e","added_by":"auto","created_at":"2025-07-21 13:44:08","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12566,"visible":true,"origin":"","legend":"Dataset 2","description":"","filename":"dataLHTfecundityhatchratelabSuppFig1.csv","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/fbbfa8d629186f9c5f41e73b.csv"},{"id":87203801,"identity":"4d85e34f-8853-4681-b719-c66fd4c50f5e","added_by":"auto","created_at":"2025-07-21 13:44:08","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12265,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"dataCIcrosses.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/893e9f471294e6b5519117b2.xlsx"},{"id":87206229,"identity":"56aaf91c-b8d8-4e65-b772-614ff5db9c38","added_by":"auto","created_at":"2025-07-21 14:08:08","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1402,"visible":true,"origin":"","legend":"Dataset 4","description":"","filename":"dataLHTmalecompetitivenesslabSuppFig1.csv","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/8bd995c5416f10b009a38b9d.csv"},{"id":87205657,"identity":"ae38e839-6b94-486a-afbd-8e1044de2197","added_by":"auto","created_at":"2025-07-21 14:00:08","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10168,"visible":true,"origin":"","legend":"Dataset 7","description":"","filename":"datawPipfemalecontaminationreleasesiteFig4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/5702858ec1e12f1967084b49.xlsx"},{"id":87204389,"identity":"72c1e150-9418-4852-9438-8f95bc72cae6","added_by":"auto","created_at":"2025-07-21 13:52:08","extension":"csv","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":232710,"visible":true,"origin":"","legend":"Dataset 6","description":"","filename":"datanumberhatchedeggshatchrateFig2.csv","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/2effe8e5d0e1286fb70cac29.csv"},{"id":87203808,"identity":"9a834388-5b96-4e22-a451-1e82f9c47395","added_by":"auto","created_at":"2025-07-21 13:44:08","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":19064,"visible":true,"origin":"","legend":"Dataset 5","description":"","filename":"dataMRR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/ed22406d3f11f1daebe8a58a.xlsx"},{"id":87205660,"identity":"db277f6d-eabc-4d79-b737-ac9fbaf7cedb","added_by":"auto","created_at":"2025-07-21 14:00:08","extension":"csv","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":36019,"visible":true,"origin":"","legend":"Dataset 3","description":"","filename":"dataLHTlongevitylabSuppFig1.csv","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/98c398bdff03f9d4d6d18443.csv"},{"id":87203828,"identity":"7d60c88b-5769-4fe4-90cc-b8b37af8b0f8","added_by":"auto","created_at":"2025-07-21 13:44:08","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":20418666,"visible":true,"origin":"","legend":"Supplementary figures","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6936662/v1/1029cfde3d0dcb68c765d053.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Production and shipment of Wolbachia-infected eggs allow controlling Aedes albopictus through the Incompatible Insect Technique on a remote island","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAedes albopictus\u003c/em\u003e has for a long time been considered as a poor disease vector because of a zoophilic behaviour and a limited competence for the transmission of some arboviruses\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This mosquito species is being increasingly scrutinized because it has been the major vector of a number of dengue or chikungunya epidemics in tropical regions within the last decades. Most importantly, the plasticity and invasive behaviour of this vector together with accelerating global changes are leading to a rapid expansion of its geographic distribution, including in temperate countries where \u003cem\u003eAe. albopictus\u003c/em\u003e has proven efficient for autochthonous transmission of Dengue virus\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In the absence of available vaccines, transmission is routinely reduced by controlling vector populations using insecticides. However, more environmental-friendly methods, such as the Sterile Insect Technique (SIT) or the Incompatible Insect Technique (IIT) are being developed to bypass the selection of insecticide resistance and reduce the impacts of pesticides on non-target species (Dobson, 2021). Both methods rely on the release of sterile males to reduce the population size of the targeted species, offering the advantage of species-specific control allowing sterilization of females in cryptic habitats or effective control of insecticide-resistant mosquito populations\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIIT leverages the properties of \u003cem\u003eWolbachia pipientis\u003c/em\u003e, a maternally inherited endosymbiotic bacterium that infects approximately 40% of arthropod species\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eWolbachia\u003c/em\u003e can manipulate mosquito reproduction through Cytoplasmic Incompatibility (CI), a form of conditional embryonic lethality that occurs when a \u003cem\u003eWolbachia\u003c/em\u003e infected male mates with a female that is either uninfected or infected by a different, incompatible \u003cem\u003eWolbachia\u003c/em\u003e strain\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. CI can be understood as resulting from a modification/rescue mechanism (\u003cem\u003emod\u003c/em\u003e/\u003cem\u003eresc\u003c/em\u003e), wherein the \u003cem\u003emod\u003c/em\u003e function modifies the sperm during spermatogenesis and the \u003cem\u003eresc\u003c/em\u003e function, expressed in the egg cytoplasm laid by \u003cem\u003eWolbachia\u003c/em\u003e-infected females, rescues the embryo through an interaction with the modified sperm\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCI can be hijacked for the development of the IIT, which involves the release of \u003cem\u003eWolbachia\u003c/em\u003e- infected males that are incompatible with resident females\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Recent studies have demonstrated the effectiveness of standalone IIT in controlling \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003eaegypti\u003c/em\u003e populations in the U.S\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, Australia\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, Singapore\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and Puerto Rico\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e as well as \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e populations in China\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Importantly, all \u003cem\u003eWolbachia\u003c/em\u003e-transinfected lines used in the aforementioned field assays exhibit unidirectional CI with the resident females, meaning that accidentally released females are compatible with resident males, thus increasing the risk of population replacement with \u003cem\u003eWolbachia\u003c/em\u003e transinfected mosquitoes. To mitigate this risk, which may lead to IIT failure, it has been proposed to combine incompatible and sterile insect techniques, utilizing \u003cem\u003eWolbachia\u003c/em\u003e infection to induce male sterility and a low irradiation dose to sterilize those females that the sexing processes fail to eliminate\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. An alternative approach is the exploitation of a bidirectional pattern of CI (Bi-CI), occuring when the native \u003cem\u003eWolbachia\u003c/em\u003e infection is removed and replaced by a foreign \u003cem\u003eWolbachia\u003c/em\u003e inducing CI\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In this case, transinfected females are only compatible with co-released transinfected males, significantly reducing the invasive potential of \u003cem\u003eWolbachia\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Indeed, assuming a similar fitness of natural and \u003cem\u003eWolbachia\u003c/em\u003e transinfected mosquitoes, \u003cem\u003eWolbachia\u003c/em\u003e infection frequency must exceed 50% to allow invasion\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, compared to \u0026gt;\u0026thinsp;0% in the case of uni-CI. Under these circumstances, population replacement could occur only if the objective of IIT deployment is complete elimination, which to our knowledge has never been achieved in any SIT/IIT mosquito program\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. When Bi-CI occurs, models suggest that immigration rates of approximately 2% of wild type individuals in the release area would be sufficient to mitigate the risk of population replacement in the long term\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Thus, the use of standalone IIT with a \u003cem\u003eWolbachia\u003c/em\u003e strain inducing Bi-CI is expected to achieve high levels of population suppression with limited risk of population replacement. However, this approach has been discussed primarily in theoretical terms\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and no field trials have yet demonstrated sustained, high-level suppression using stable bi-directional CI over the long term.\u003c/p\u003e \u003cp\u003eIn this context, we implemented a six-month IIT program aiming at controlling an \u003cem\u003eAedes albopictus\u003c/em\u003e population on a 9-hectare island in the Seychelles, utilizing a mono-transinfected mosquito line that induces bi-directional CI with the local population. We demonstrate that IIT deployment achieves population suppression reaching 95% within three months of the initial release, and a suppression level maintained above 50% until the end of the release period. Moreover, we show that while achieving a high suppression level over several months, the contamination percentage of incompatible individuals in the field never exceeds 7% and drops to 0% two months after the end of the releases. Finally, we demonstrate the feasibility of a mass production model in which mosquito eggs are produced at a central facility and subsequently distributed to a satellite rearing center dedicated to producing incompatible males only\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Altogether these results support the viability of this mass production model for industrial deployment.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCI penetrance of the SEY-\u003c/b\u003e \u003cb\u003ew\u003c/b\u003e \u003cb\u003ePip line\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIncompatible male releases were conducted using an \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus w\u003c/em\u003ePip transinfected line (Scussel et al., 2024) introgressed with a wild genetic background from the Seychelles, resulting in the SEY-\u003cem\u003ew\u003c/em\u003ePip line. Reciprocal crosses between wild lines and SEY-\u003cem\u003ew\u003c/em\u003ePip confirmed that \u003cem\u003ew\u003c/em\u003ePip induced bidirectional CI in \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e regardless of the host genetic background\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Indeed, when SEY-\u003cem\u003ew\u003c/em\u003ePip males were crossed with wild females, they induced nearly complete CI, with a hatching rate ranging from 0.37 to 1.05%. Symmetrically, when wild males were crossed with SEY-\u003cem\u003ew\u003c/em\u003ePip females, the hatching rate ranged from 0.47 to 9,41% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHatch rate obtained in control and reciprocal crosses between the SEY-\u003cem\u003ew\u003c/em\u003ePip and three different wild lines.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecrosses\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003enb of hatched\u003c/p\u003e \u003cp\u003eeggs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003enb of unhatched eggs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehatch rate (%) (\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCI (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCIcorr (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e♂ SEY-\u003cem\u003ew\u003c/em\u003ePip x ♀ Le Port\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9,053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1,05 (\u0026plusmn;\u0026thinsp;1,67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98,95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e92,93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e♂ SEY-\u003cem\u003ew\u003c/em\u003ePip x ♀ S-RUN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8,007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,59 (\u0026plusmn;\u0026thinsp;0,70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99,41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97,38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e♂ SEY-\u003cem\u003ew\u003c/em\u003ePip x ♀ La Providence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4,683\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,37 (\u0026plusmn;\u0026thinsp;0,07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99,63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e98,33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e♂ Le Port x ♀ SEY-\u003cem\u003ew\u003c/em\u003ePip\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7,491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,76 (\u0026plusmn;\u0026thinsp;0,31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99,24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e96,34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e♂ S-RUN x ♀ SEY-\u003cem\u003ew\u003c/em\u003ePip\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5,401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0,47 (\u0026plusmn;\u0026thinsp;0,21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99,53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97,74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e♂ La Providence x ♀ SEY-\u003cem\u003ew\u003c/em\u003ePip\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4,562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9,41 (\u0026plusmn;\u0026thinsp;2,96)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90,59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e54,69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e♂ Le Port x ♀ Le Port\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10,468\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,701\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e85,14 (\u0026plusmn;\u0026thinsp;7,30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e♂ S-RUN x ♀ S-RUN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5,416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1,652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e77,51 (\u0026plusmn;\u0026thinsp;4,92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e♂ La Providence x ♀ La providence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1,918\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e563\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e77,88 (\u0026plusmn;\u0026thinsp;5,80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e♂ SEY-\u003cem\u003ew\u003c/em\u003ePip x ♀ SEY-\u003cem\u003ew\u003c/em\u003ePip\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9,875\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2,483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e79,23 (\u0026plusmn;\u0026thinsp;7,94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLife history traits (LHTs) and mating competitiveness of the SEY-\u003c/b\u003e \u003cb\u003ew\u003c/b\u003e \u003cb\u003ePip line in laboratory conditions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess the fitness of the SEY-\u003cem\u003ew\u003c/em\u003ePip line following introgression, we compared different LHTs with those of two wild lines, S-RUN and Le Port. The traits analyzed included longevity, number of laid eggs and hatching rate. For both sexes, Le Port line exhibited greater longevity than the S-RUN and SEY-\u003cem\u003ew\u003c/em\u003ePip lines (log-rank test; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for both comparisons), and SEY-\u003cem\u003ew\u003c/em\u003ePip also had a longer longevity than S-RUN, although this difference was significant in males only (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). We also observed a significant difference in the number of laid eggs between Le Port line, recording an average of 47.25 (\u0026plusmn;\u0026thinsp;14.65) eggs laid per female, and both SEY-\u003cem\u003ew\u003c/em\u003ePip and S-RUN lines, for which similar number of eggs were laid, 40.52 (\u0026plusmn;\u0026thinsp;12.30) and 42.19 (\u0026plusmn;\u0026thinsp;16.01), respectively (GLMM; quasi-poisson family; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.627) (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We observed a similar hatch rates in Le Port and S-RUN lines, with a mean of 83.97% (\u0026plusmn;\u0026thinsp;19.25) and 82.94% (\u0026plusmn;\u0026thinsp;21.05), respectively. These hatch rates were significantly higher than those observed for the SEY-\u003cem\u003ew\u003c/em\u003ePip line showing a mean value of 75.89% (\u0026plusmn;\u0026thinsp;25.75) (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eFinally, we assessed the mating competitiveness of SEY-\u003cem\u003ew\u003c/em\u003ePip males compared to wild-type males from the S-RUN and Le Port lines, by crossing virgin wild females with increasing ratios of incompatible (SEY-\u003cem\u003ew\u003c/em\u003ePip) to compatible (S-RUN or Le Port) males. Observed hatch rates at 1 : 0 and 0 : 1 ratios (where the first and second numbers correspond to the proportions of compatible and incompatible males, respectively) were compared with the expected hatch rates under the assumption that both male types had equal mating competitiveness. As expected, hatch rates decreased with increasing proportions of incompatible males, in crosses involving wild females from S-RUN or Le Port lines (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Of note, when different combinations of SEY-\u003cem\u003ew\u003c/em\u003ePip and S-RUN males were used, observed hatch rates were consistently and significantly lower than expected, indicating that incompatible males were more competitive than S-RUN males. In crosses involving SEY-\u003cem\u003ew\u003c/em\u003ePip and Le Port males, observed and expected hatch rates were not different at 1:5 and 1:10 ratios. However, at a 1:1 ratio, the observed hatch rate was 20% higher than expected. Altogether, SEY-\u003cem\u003ew\u003c/em\u003ePip appears as a mono-transinfected line suitable for mass-production and subsequent use in an IIT program, as the potential fitness cost associated with \u003cem\u003ew\u003c/em\u003ePip infection and/or genetic introgression are absent or minimal.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of the natural population size before releases\u003c/h2\u003e \u003cp\u003eThe density of mosquito population at the release site was evaluated using mark-release-recapture (MRR) experiments. A total of 4,389 and 5,682 marked males were released during the first and second MRR experiment, conducted in October and November 2022, respectively performed 9 and 8 months before the first release. For both MRRs, the recapture rate of marked males was 1.5% over four consecutive days (68/4389 for the first and 87/5682 for the second MRR). Based on the first MRR, we estimated a total number of wild males on the whole island of 9,994 (\u0026plusmn;\u0026thinsp;1,984), while the second yielded an estimate of 15,540 (\u0026plusmn;\u0026thinsp;3743). We used the data of the second MRR to calibrate the number of incompatible males to be released to ensure a minimum incompatible male ratio of 5:1. This number was estimated to ~\u0026thinsp;80,000 males per week (15,540*5).\u003c/p\u003e \u003cp\u003e \u003cb\u003eShipping of eggs, release of incompatible males and suppression of the\u003c/b\u003e \u003cb\u003eAe\u003c/b\u003e. \u003cb\u003ealbopictus\u003c/b\u003e \u003cb\u003epopulation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe control site consists of a 7-hectare area in the Southern part of Saint Anne island, while the release site encompassed the entire 9-hectare area of Moyenne island. These sites are separated by 1.2 kilometers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mosquito population dynamics in both sites was monitored weekly before male releases, using ovitraps from 15th February, 2022 to 27th June, 2023. During this period, population trends were similar (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), as supported by the absence of significant difference in the number of hatched eggs between the two sites (Wilcoxon rank sum test; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.608), and confirming the suitability of the selected control site. Likewise, no significant difference was observed in egg hatching rates between the release (95.27\u0026thinsp;\u0026plusmn;\u0026thinsp;5.09%) and control (94.87\u0026thinsp;\u0026plusmn;\u0026thinsp;5.79%) sites during the prerelease phase (GLMM-quasi-binomial family; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.947) (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThree months before the start of incompatible male releases, eggs of the SEY-\u003cem\u003ew\u003c/em\u003ePip line, continuously produced in Saint-Denis (La R\u0026eacute;union) were shipped weekly to the Seychelles in bubble wraps via commercial airlines. Over nine months of shipping, a total of approximately 145g of eggs (~\u0026thinsp;4 g per week) was transported. Delivery time ranged from 7 to 15 days, depending on the regulatory inspection process at the Seychelles airport. Eggs were sent out in 32 shipments with an overall sending success rate of 90% (29/32), meaning that the observed hatching rate was close to the expected value.\u003c/p\u003e \u003cp\u003eReleases of incompatible males started on 13th July 2023, and were maintained for six consecutive months. The release period was eventually separated in three phases, with distinct numbers of released males (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Approximately 80,000 males were released every week for twelve weeks during phase I. We observed an immediate suppression in the first week following the first release, with a 30% decrease in the hatch rate at the release site. By the second week, the hatch rate had dropped by 60%. At that time, we measured a male release ratio of 23:1 (SEY-\u003cem\u003ew\u003c/em\u003ePip to wild males). Then the hatch rate continued to decline throughout phase I, reaching 18.65% at the end of phase I (three months after the first release), compared to 93.10% in the control site (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Although the number of hatched eggs temporarily decreased in the control site during the first few weeks after the first release, it remained significantly lower in the release site throughout phase I (13th July- 29th September, 2023) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The impact of IIT deployment became particularly obvious from the eighth week following the first release, even if the male release ratio dropped to 1.5:1 (SEY-\u003cem\u003ew\u003c/em\u003ePip to wild males). At this point, the average number of hatched eggs in the control site began to increase weekly, reaching a mean of 49.15 (\u0026plusmn;\u0026thinsp;33.92) by the end of phase I. In contrast, the number of hatched eggs remained consistently low in the release site, with a mean of 3.08 (\u0026plusmn;\u0026thinsp;8.27) during the same period. Additionally, at the end of the phase I, 12 out of 26 ovitraps in the release site contained no hatched eggs. At this point the male release ratio increased to 7:1 (SEY-\u003cem\u003ew\u003c/em\u003ePip to wild males). After twelve weeks of releases, this resulted in an overall population suppression level of 95% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter achieving a population suppression of 95%, we reduced the number of incompatible males released to approximately 50,000 per week, for six consecutive weeks, corresponding to phase II (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). During this phase, we were able to maintain control over the population size, as evidenced by a significant difference in the number of hatch eggs between the release and the control sites (Wilcoxon rank sum test; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, towards the end of phase II, we observed an increase in the number of hatched eggs in the release site during the final three weeks, coinciding with a lower male release ratio of 1:1 (SEY-\u003cem\u003ew\u003c/em\u003ePip to wild males). Between the end of phase I and phase II the hatch rate increased by 23%, while the suppression level decreased by 35% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C), with a male release ratio remaining at 1:1 (SEY-\u003cem\u003ew\u003c/em\u003ePip to wild males) by the end of phase II.\u003c/p\u003e \u003cp\u003eIn response to the decline in effectiveness, we increased the number of incompatible males released to approximately 140,000 per week for the last four weeks of releases, corresponding to phase III. This adjustment significantly restored control efficiency, stabilizing the hatch rate and increasing the population suppression level, which reached 80% by the end of releases. During phase III, we also observed a significant difference in the number of hatched eggs between the release and control sites (Wilcoxon rank sum test; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eTo investigate the duration of suppression following the end of releases, we continued to monitor the population for ten additional weeks. During this post-release survey, we observed a significant difference in the number of hatched eggs between sites (Wilcoxon rank sum test; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, starting from the seventh week after the last release, the hatch rate and the number of hatched eggs in the release site became comparable to those observed in the control site.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSpatial heterogeneity of mosquito suppression\u003c/h3\u003e\n\u003cp\u003eAn analysis of spatial population dynamics revealed that egg hatch was impacted by the release of incompatible males at all surveyed ovitraps. However, we found that this impact was heterogeneous, with some areas being more difficult to maintain at low densities throughout the entire release period, particularly in the southwestern part of the release site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAbsence of population replacement\u003c/h3\u003e\n\u003cp\u003eThe experimental set-up allowed addressing whether standalone IIT using a transinfected line exhibiting bi-CI could strongly suppress a field population without causing population replacement. To assess this, we firstly quantified weekly the female contamination rate in the male pupae production following mechanical sex separation. The female contamination rate ranged from 0.015 to 0.08% throughout the entire mass production period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). During this quality control of the production, all pupae identified as females were removed. However, since not all male pupae were verified and because human verification is inherently imperfect, the release of accidental females did occur as shown by the presence of \u003cem\u003ew\u003c/em\u003ePip females at the study site (see below).\u003c/p\u003e \u003cp\u003eThus, to monitor the risk of SEY-\u003cem\u003ew\u003c/em\u003ePip mosquito establishment in the field, we collected and reared all larvae from eggs found in ovitraps at the release site every three weeks, from phase I through the end of the post-release phase. Larvae were reared until adulthood to determine the percentage of \u003cem\u003ew\u003c/em\u003ePip-positive individuals per ovitrap. In total, this monitoring was conducted seven times during the releases and four times during the post-release period, representing 1,196 screened adults.\u003c/p\u003e \u003cp\u003eWe observed a \u003cem\u003ew\u003c/em\u003ePip-positive rate ranging from 0 to 6.3% during the release period, with a maximum of three positive ovitraps (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Although \u003cem\u003ew\u003c/em\u003ePip-positive individuals were detected several weeks after the last release, their frequency never exceeded 7%. Importantly, no positive samples (=\u0026thinsp;176) were found in the final screening conducted more than two months after the last release. Moreover, analysis of the spatial distribution of positive ovitraps revealed that they were located in different areas across screening, with only one ovitrap testing positive on two consecutive occasions (Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings support the hypothesis of an absence of local establishment and instead suggest sporadic releases, involving one or a few females released at different locations from week to week.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eAedes albopictus\u003c/em\u003e is playing a major role in the emergence or re-emergence of dengue and chikungunya in tropical regions and has also been implicated in outbreaks in newly-colonized temperate regions such as Italy in 2007\u003csup\u003e29\u0026ndash;32\u003c/sup\u003e. Although proof-of-concept examples remain limited, the Incompatible Insect Technique (IIT) and the Sterile Insect Technique (SIT) have already demonstrated their effectiveness in controlling \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e populations (Aldridge, Gibson, \u0026amp; Linthicum, 2024).\u003c/p\u003e \u003cp\u003eOne of the main advantages of IIT over SIT is that males are \u0026ldquo;ready to use\u0026rdquo;. In SIT programs, the rearing facility producing sterilized insects must be located near the release sites, or sterile males must be transported over long distances, in which case handling conditions (such as chilling and compaction) need to be optimized and transport minimized to prevent adverse effects on males\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. However, SIT offers the sustained advantage over IIT that accidentally released female are sterile. This limitation of IIT is particularly salient in the case of uni-directional CI, which to our knowledge has been thus far used in all long-term published IIT studies aiming at suppressing \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e populations.\u003c/p\u003e \u003cp\u003eTo overcome this limitation, we implemented a long term standalone IIT program for the control of \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e using a transinfected line exhibiting bi-CI with the local population. We conducted this trial in \u0026ldquo;real-life\u0026rdquo; conditions, meaning through the monitoring of mosquito dynamics during suppression and subsequent adjustment of release conditions. The release of SEY-\u003cem\u003ew\u003c/em\u003ePip males led to a 95% population suppression within only twelve weeks. This level of suppression was reached although the estimated male release ratio was low, 1.5:1 and 7:1 (SEY-\u003cem\u003ew\u003c/em\u003ePip to wild males), 8 and 12 weeks after the first release, respectively. These results suggest that the mono-transinfected line used in this study allowed efficient suppression even when using low incompatible to wild male ratios. Such a performance is in keeping with previously published data showing that the introgression of a genetic background from the targeted population does limit fitness costs associated with \u003cem\u003eWolbachia\u003c/em\u003e transinfection\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter achieving a substantial level of population suppression (phase I), we choose to decrease the number of released males from ~\u0026thinsp;80,000 to 50,000 per week (phase II). Although at this time the number of wild mosquitoes was low due to efficient suppression, the number of hatching eggs rapidly increased as exemplified by two consecutive measures following releases with low SEY-\u003cem\u003ew\u003c/em\u003ePip to wild males ratio (1:1), potentially due to favorable climatic conditions offered by the onset of the rainy season.\u003c/p\u003e \u003cp\u003eFinally, we increased the number of incompatible males to ~\u0026thinsp;140,000 per week (phase III) for the last three releases, allowing restoring 80% suppression. Altogether, these data indicate that, despite a significant reduction of the wild population size, a minimal incompatible male ratio should be conserved for a long term population control.\u003c/p\u003e \u003cp\u003eInterestingly, we showed that the suppression effect was heterogeneous at the release site, potentially resulting from habitat heterogeneity, as reported in other IIT program\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. We observed that ovitrap M01, located in the southwest part of the release site, displayed consistently lower suppression than surrounding ovitraps. We hypothesized that this difference resulted from favorable breeding conditions around this trap, neighboring a bamboo area (where cut bamboo stems provide larval sites unless they are refilled). This interesting pattern suggests that constant monitoring of the treated areas should be associated with local tuning of the number of released males, with increasing numbers being used in areas identified as resisting to mosquito suppression.\u003c/p\u003e \u003cp\u003eWhile presented data show the effectiveness of a standalone IIT for the control of an \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e population in an insular context, we demonstrate that a significant population suppression can be achieved without population replacement. Indeed, although we detected \u003cem\u003ew\u003c/em\u003ePip-positive individuals during and shortly after the release period (ranging from 0 to 6.3%), no incompatible individuals were found in our final screening conducted two months after the last release. We acknowledge that visual inspection of a large number of pupae performed in this study is time consuming and can be hardly implemented at substantially larger scales. To overcome this limitation, it has been proposed to implement an individual automated verification at the adult stage allowing to reduce drastically the female contamination rate\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Classically, female pupae contamination after classic mechanical sex separation ranges from 0.5 to 3%\u003csup\u003e11,38\u003c/sup\u003e. Here we demonstrated that female contamination can be reduced to 0.02%, collecting pupae on two consecutive days for each production batch.\u003c/p\u003e \u003cp\u003eLastly, this program was carried out on a remote island, representing one of the most challenging scenarios for the deployment of IIT as population replacement cannot be mitigated by the migration of wild mosquitoes surrounding the treated area. Presented data highlight another advantage of the deployment of a standalone IIT: the possibility of developing a mass production model based on a unique central facility for eggs production associated with satellite units dedicated to the production of incompatible males. Such satellite units are relatively easy to set-up as there is little need for space and labor as equipments for blood-feeding or egg collection are not unnecessary\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. As compared to the shipping of chilled adults from a central facility to satellite units, the shipping of eggs, facilitated by the resistance of \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e eggs to desiccation and their low weight, ensures the production of high quality males while avoiding a quality control on males post-shipping. This model of mass production can also be developed in SIT programs under the strict condition that an irradiator is present at each satellite unit, which is clearly challenging but should be considered in the case of a mosquito extinction program.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eMosquito lines\u003c/h2\u003e\n \u003cp\u003eWe firstly constructed a transinfected line with a wild genetic background from the Seychelles. For this, we used a laboratory line transinfected with \u003cem\u003ew\u003c/em\u003ePip-IV\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, a \u003cem\u003eWolbachia\u003c/em\u003e strain naturally found in \u003cem\u003eCulex pipiens\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Females from this line were backcrossed with males from a wild line sampled using ovitraps on Ile Longue (Seychelles), located about 400 meters from the release site. Backcrossing was performed using 15 day-old wild males (using the F\u003csub\u003e1\u003c/sub\u003e post sampling) with 4\u0026ndash;5 day-old virgin females from the transinfected line for four consecutive generations, resulting in a line named SEY-\u003cem\u003ew\u003c/em\u003ePip, expected to display 93,75% of the wild nuclear genetic background.\u003c/p\u003e\n \u003cp\u003eWe used three other wild lines, named S-RUN, Le Port, and La Providence, to conduct laboratory experiments aimed at quantifying CI level induced by the SEY-\u003cem\u003ew\u003c/em\u003ePip line and at measuring possible fitness costs following introgression. The S-Run and Le Port lines were originally derived from a natural population on Reunion Island\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and were maintained for 50 and 10 generations, respectively, before experimentation. The La Providence line was sampled from the Providence neighborhood on Mah\u0026eacute; island (the largest island in the Seychelles archipelago) and maintained for 7 generations before experiments.\u003c/p\u003e\n \u003cp\u003eAdults of all lines were maintained at a temperature of 27\u0026thinsp;\u0026plusmn;\u0026thinsp;1◦C, a relative humidity of 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5% and a 12 : 12 h light:dark photoperiod in the insectary located at Saint-Denis (Reunion island). Females were blood-fed using the Hemotek system (Hemotek Ltd, United Kingdom), bovine blood provided by the regional slaughterhouse (Saint-Pierre, Reunion island) and supplemented with EDTA (0,1%).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eCI expression of the transinfected line\u003c/h3\u003e\n\u003cp\u003eTo quantify CI penetrance of the SEY-\u003cem\u003ew\u003c/em\u003ePip line following introgression, we performed reciprocal \u003cem\u003een masse\u003c/em\u003e crosses using S-RUN, Le Port and La Providence lines. Although \u003cem\u003een masse\u003c/em\u003e crosses can mask individual variability, especially in the case of moderate CI, complete or nearly complete CI were expected\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, which led us to favor \u003cem\u003een masse\u003c/em\u003e rather than individual crosses in order to screen a larger number of mosquitoes. Crosses involved 2-5-day-old virgin females and males (100 from each sex) in 15 \u0026times; 15 \u0026times; 15 cm cages (Bugdorm, Taiwan). Three replicates were performed for each cross. Females were given a blood meal 48h after mating and eggs were collected 5 days later. After 7 days of drying, eggs were allowed to hatch for 24 h (in a jar containing 250 mL tap water supplemented with 50 mg of TetraMin (TETRA) and the number of hatched and unhatched eggs was counted. A hatch rate was calculated as follows: hatch rate = (number of hatched eggs/total number of eggs) \u0026times; 100. To account for the embryonic mortality not related to CI, we used a corrected CI index (CI\u003csub\u003ecorr\u003c/sub\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e calculated as follows: CI\u003csub\u003ecorr\u003c/sub\u003e = [(CI\u003csub\u003eobs\u003c/sub\u003e \u0026minus; CCM)/(100\u0026thinsp;\u0026minus;\u0026thinsp;CCM)] \u0026times; 100, where CI\u003csub\u003eobs\u003c/sub\u003e is the percentage of unhatched eggs observed in a given incompatible cross, and CCM is the mean mortality observed in the control crosses.\u003c/p\u003e\n\u003ch3\u003eLife history traits of the transinfected line\u003c/h3\u003e\n\u003cp\u003eLongevity and fecundity of the SEY-\u003cem\u003ew\u003c/em\u003ePip line were compared to those of the S-RUN and Le Port lines, both showing high fitness under laboratory-controlled conditions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Larvae-rearing conditions were standardized between lines for all experiments. Specifically, eggs from each line were allowed to hatch for 24 h at 31\u0026deg;C in containers containing 250 ml of water supplemented with 50 mg TetraMin (TETRA). For each line, 2,500 L1 were manually counted and transferred to a tray (53 \u0026times; 325 \u0026times; 65 cm, MORI 2A) and fed with a controlled quantity of food (TetraMin (TETRA), day 1: 0.45 g, day 2: 1 g, day 3: 1.25 g, day 4: 1 g, day 5: 1 g, day 6: 0.75 g, day 7: 0.5 g) until pupal stage. Male and female pupae were initially separated using a pupae sex sorter (Wolbaki, WBK-P0001-V1 model), and then individually inspected under a binocular loupe.\u003c/p\u003e\n\u003cp\u003eFor each line, longevity was measured by introducing 100 newly emerged males or females separately in 15 \u0026times; 15 \u0026times; 15 cm cages (Bugdorm, Taiwan) in which sugar meal (5%) was changed weekly. Three replicates were performed at different times for each line and sex and longevity was determined by recording the number of dead mosquitoes for 45 days.\u003c/p\u003e\n\u003cp\u003eFor each line, the number of laid eggs was measured by placing 200 male and 200 female pupae (one cage per line) in 30 \u0026times; 30 \u0026times; 30 cm cages (Bugdorm, Taiwan) and left for 3 days following emergence. A blood meal was then provided and engorged females were placed in a separate cage. Five days later, 50 females were randomly selected and placed individually in a small plastic cup for egg laying. Cups with at least one laid egg were conserved for the analyses. After 7 days of drying, eggs were counted, allowed to hatch for 24 h and hatch rates were measured. Three replicates were performed at different times for each line.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eMating competitiveness of incompatible males in laboratory-controlled conditions\u003c/h2\u003e\n \u003cp\u003eWe evaluated the mating competitiveness of incompatible males by mixing virgin females from the S-RUN or Le Port line with increasing ratios of males from the SEY-\u003cem\u003ew\u003c/em\u003ePip line. Pupae from both lines were allowed to emerge in separate 30 \u0026times; 30 \u0026times; 30 cm cages (Bugdorm, Taiwan) and adults were provided sugar meal (5%). Mating competitiveness was monitored using 2-3-day old virgin females and males. A hundred males were first placed inside cages followed by the release of an equal number of females, and mating was allowed for 48 h. All males were then removed, a blood meal was provided to females and eggs were collected by oviposition \u003cem\u003een masse\u003c/em\u003e 5 days later. After 7 days of drying, eggs were allowed to hatch for 24 h and the hatching rate was measured and used as a proxy of mating competitiveness. Five ratios of males were tested: 1 : 1 (50♂ S-RUN or Le Port : 50♂ SEY-\u003cem\u003ew\u003c/em\u003ePip), 1 : 5 (17♂ S-RUN or Le Port : 85♂ SEY-\u003cem\u003ew\u003c/em\u003ePip), 1 : 10 (9♂ S-RUN or Le Port: 90♂ SEY-\u003cem\u003ew\u003c/em\u003ePip) and two control ratios, 1 : 0 (100♂ S-RUN or Le Port: 0♂ SEY-\u003cem\u003ew\u003c/em\u003ePip) and 0 : 1 (0♂ S-RUN or Le Port: 100 SEY-\u003cem\u003ew\u003c/em\u003ePip). Three replicates in different times were performed for each cross and ratio.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eDescription of study areas\u003c/h2\u003e\n \u003cp\u003eBoth study sites were located within the Sainte Anne Marine National Park in the Seychelles. The control site consists of a 7-hectare area in the Southern part of Saint Anne island, while the release site encompassed the entire 9-hectare area of Moyenne island (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). These sites, separated by 1.2 kilometers, presented comparable ecological conditions, with similar vegetation and geology, providing suitable climatic conditions for \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e populations. The region is classified as having a tropical maritime climate, with year-round temperatures ranging from 22\u0026deg;C to 33\u0026deg;C. The rainy season extends from November to April, peaking in January and February, while the dry season lasts from May to October. Relative humidity is generally high, often exceeding 80%. Experiment approvals were provided by the Seychelles Bureau of Standards (SBS) and the National Biosecurity Agency (NBA), in accordance with local regulations on insect population release and monitoring.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEstimating\u003c/strong\u003e \u003cstrong\u003eAe\u003c/strong\u003e. \u003cstrong\u003ealbopictus\u003c/strong\u003e \u003cstrong\u003enatural population size at the release site\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe natural population size of \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e at the release site was estimated through two mark-release-recapture (MRR) experiments conducted prior releases. Adults used for MRR were obtained from eggs collected in the wild via ovitraps, which were deployed to monitor the temporal dynamics of \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e population size at the release site (see below). Larvae were reared under laboratory-controlled conditions until pupal stage. Male and female pupae were initially separated using a pupae sex sorter (Wolbaki, WBK-P0001-V1 model), and all male pupae were individually inspected under a binocular loupe.\u003c/p\u003e\n \u003cp\u003eTo mark adult males with fluorescent pigment, we constructed a self-marking unit adapted from a previously described prototype\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. A total of 2,500 male pupae was placed in a square petri dish (12.5 x 12.5 cm) with approximately 0.5 cm water, which was previously positioned inside a 30 x 30 x 30 cm cage (Bugdorm-1 model) (Bugdorm, Taiwan). A removable exit grid, consisting of wool yarns lines spaced approximately 0.5 cm apart and pre-coated with fluorescent pigment (Radiant color), was then placed above the petri dish. Upon emergence, adult males were self-marked as they passed through the grid and were subsequently conserved in the cage with a sugar solution 5% until release. The self-marking unit was then removed from the cage, and the number of males that had not crossed the grid was counted. Just before the release, dead adults were removed from the cage to ensure an accurate count of the alive marked males being released.\u003c/p\u003e\n \u003cp\u003eAltogether, 4,389 and 5,682 marked males were released during the first and second MRR experiments, conducted in October and November 2023, respectively. For both MRRs, males were released at four equidistant spots, surrounded by 27 BG traps (Supp. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), corresponding to an area of ~\u0026thinsp;4.5 ha. Just after releases, each BG trap was activated for four days of capture, with a collection bag being replaced every 24 h. All captured individuals were frozen at -20\u0026deg;C for 24h, species and sex of each trapped adult was determined under a binocular loupe. All \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e males were then examined under ultraviolet light to detect the presence of fluorescent dust. The population size was estimated as follows: \u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003en\u003c/em\u003e * (\u003cem\u003eM\u003c/em\u003e/\u003cem\u003em\u003c/em\u003e), where \u003cem\u003eN\u003c/em\u003e is the population size per day, \u003cem\u003en\u003c/em\u003e the number of captured mosquitos, \u003cem\u003eM\u003c/em\u003e the number of released marked mosquitos, and \u003cem\u003em\u003c/em\u003e the number of recaptured marked mosquitos\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eMass Production and release of incompatible males\u003c/h2\u003e\n \u003cp\u003eMass production and subsequent release of SEY-\u003cem\u003ew\u003c/em\u003ePip males involved five steps: (i) egg production at the CYROI insectary (Saint-Denis, Reunion island), (ii) transportation of eggs to Seychelles using a commercial airline company, (iii) larvae rearing and sex separation at insectary of the Ministry of Health of Seychelles (Grand Anse, Seychelles), (iv) transportation of male pupae by boat to the release site, and (v) release of adult males.\u003c/p\u003e\n \u003cp\u003eFor egg production, we maintained adult cages (45\u0026times;45\u0026times;45 cm) (Bugdorm, Taiwan) continuously, each containing approximately 6,000 female pupae and 2,000 male pupae. Adults were fed with a 5% sugar solution. Females were blood-fed three times a week with bovine blood, and eggs were collected weekly for three consecutive weeks. Eggs were matured for one week before being used either for the production of adults used for egg production or sent to Seychelles for the production of incompatible males. The procedures for egg hatching, larvae rearing and sex separation were similar in both distant insectaries. Briefly, the eggs were brushed and transferred in 120 ml plastic containers with a hatching solution (tap water with TetraMin (TETRA) at 1g/L) and left for 8 h. Approximately 3,000 larvae were added to each tray (53 \u0026times; 325 \u0026times; 65 cm, MORI 2A) and fed with a controlled quantity of food (TetraMin (TETRA), day 1: 0.5 g, day 2: 1 g, day 3: 1.25 g, day 4: 1.5 g, day 5: 0.75 g, day 6: 1 g, day 7: 0.75 g, day 8: 0.5 g) until pupal stage. Male and female pupae were separated using a pupae sex sorter (Wolbaki, WBK-P0001-V1 model).\u003c/p\u003e\n \u003cp\u003eDuring the release phase, after the initial mechanical sex separation, quality control was performed daily on all (or nearly all, depending on the production phase; see below) collected pupae considered as male pupae, by measuring female contamination under a binocular loupe.\u003c/p\u003e\n \u003cp\u003eUsing a volumetric template, daily male pupae production was divided into several lots of approximately 2,250 male pupae, placed into 500 mL containers (Nalgene) with ~\u0026thinsp;2 cm water depth. The following day, all pupae were transported by boat to the release site, and each container was transferred into a release tube (10-cm diameter x 35-cm height). The tubes were placed in trays containing\u0026thinsp;~\u0026thinsp;2 cm water depth, and a source of 10% sugar solution was placed at the top. The following day, the release tube was removed from the water and adult males were released. All male pupae produced weekly were released over two consecutive days, with the release evenly distributed across the 40 release spots, spaced approximately 40 meters apart (Supp. Figure\u0026nbsp;5).\u003c/p\u003e\n \u003cp\u003eIncompatible males were released over a period of six months, divided into three phases, each distinguished by the number of released males. Phase I involved the release of approximately 80,000 males/week over 12 weeks. Phase II involved the release of around 50,000 males/week over 6 weeks. Finally, phase III involved the release of approximately 140,000 males/week over the last four weeks (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). For phases I and II, quality control of the male pupae production after mechanical sex separation was performed on the entire production, while in phase III, quality control was conducted on only two-thirds of the production.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eMeasure of the incompatible vs. wild male ratios during the release period\u003c/h2\u003e\n \u003cp\u003eThe incompatible vs. wild male ratios were measured monthly throughout the entire release period (i.e. five measurements), from July 2023 to November 2023. For this, 9 BG-Sentinel traps (Biogents, Regensburg, Germany) were distributed across the release site, spaced approximately 90 meters apart, resulting in one trap per hectare (Supp. Figure\u0026nbsp;6). BG traps were activated shortly after each release and for 24-h of capture. Captured mosquitoes were then morphologically identified under a binocular loupe to determine sex and species of each specimen. All \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e adult males were individually placed in 1.5 ml Eppendorf tubes and stored at -20\u0026deg;C before being shipped to Reunion island for \u003cem\u003eWolbachia\u003c/em\u003e typing through PCR analysis as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eMonitoring population suppression\u003c/h2\u003e\n \u003cp\u003eTemporal dynamics of the \u003cem\u003eAe. albopictus\u003c/em\u003e population size in both the control and release sites were monitored using ovitraps before, during, and after the release periods. Ovitraps consisted of small black 800-mL buckets containing 500 mL of tap water and one egg-laying paper (Sartorius\u0026trade;) fixed with a piece of plexiglas. From February 2022 to February 2024, 26 and 20 ovitraps were distributed throughout the release and control sites, respectively, with ovitraps placed approximately 50 meters apart, resulting in approximately 3 ovitraps per hectare (Supp. Figure\u0026nbsp;7A-B). Ovitraps were retrieved and replaced weekly. Positive egg-laying papers, defined as those containing at least one egg, were preserved, and the number of eggs was recorded. Eggs were then stored at room temperature for six days, and then allowed to hatch for 24 h in a jar containing 250 mL tap water supplemented with 50 mg of TetraMin (TETRA). Hatch rate was then calculated as described previously \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eTo measure the suppression efficiency, we calculated the suppression percentage using the following equation, where Hc and Hr represent the average number of hatched eggs per trap at the control and release sites, respectively\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e:\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:SE\\left(\\text{%}\\right)=\\frac{Hc-Hr}{Hc}x100$$\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eMonitoring the risk of population replacement\u003c/h2\u003e\u003cp\u003eThe risk of population replacement at the release site, caused by accidental releases of transinfected females, was monitored from the beginning until two months after the last release (from 2 August 2023 to 20 February 2024). During this period, every three weeks, all larvae from the 26 ovitraps were conserved and reared until adult stage. Adults were then frozen for 1 h at -20\u0026deg;C, individually transferred to 1.5 mL Eppendorf tube and shipped to Reunion island laboratory facilities for PCR screening. The dynamics of \u003cem\u003ew\u003c/em\u003ePip-positive adults were monitored at eleven different time points, with a total of 1,196 individuals checked for the presence or absence of \u003cem\u003ew\u003c/em\u003ePip. Two indicators were used to monitor the risk: (i) the proportion of ovitraps containing \u003cem\u003ew\u003c/em\u003ePip-positive adults and (ii) the proportion of \u003cem\u003ew\u003c/em\u003ePip-positive individuals emerging from each positive ovitrap.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDNA extraction and\u003c/strong\u003e \u003cstrong\u003ew\u003c/strong\u003e\u003cstrong\u003ePip screening\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted from adult mosquitoes using the cetyltrimethylammonium bromure (CTAB) method\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The PCR reaction mixture consisted of 2 \u0026micro;l gDNA template, 8.5 \u0026micro;l nuclease free water, 1 \u0026micro;l of each primer (10 \u0026micro;M), and 12.5 \u0026micro;l of GoTaq\u0026reg; G2 Hot Start Master Mixes (Promega corporation). We targeted an ankirin domain protein by amplifying the \u003cem\u003eank2\u003c/em\u003e gene using the following primers: F-CTTCTTCTGTGAGTGTACGT and R2-TCCATATCGATCTACTGCGT\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. PCR amplification was performed under the following conditions: 95\u0026deg;C 5 min followed by 35 cycles of 94\u0026deg;C 45 s, 53\u0026deg;C 45 s, 72\u0026deg;C 45 s, and 72\u0026deg;C for 7 min. Amplified fragments were run in agarose gel (1.5%) electrophoresis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eLongevity data were analysed using a log-rank test. Fecundity data (count data) were analysed using a generalized linear mixed model (GLMM, quasi-poisson family, log link) in which the different mosquito lines were included as a fixed effect and the repetitions (individual egg laying) as a random factor. We used a GLMM to analyse egg hatch rate (binary data, quasi-binomial family, logit link). The overdispersion of the data was checked using a R code proposed by Ben Bolker and others (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bbolker.github.io/mixedmodels-misc/glmmFAQ.html\u003c/span\u003e\u003c/span\u003e). A F-test for the GLMM-quasi-binomial and quasi-poisson models were used to analyse deviance. Exact binomial tests were used to compare the observed and expected hatch rates in the mating competitiveness experiment (proportional data). Analyses were performed in R version 4.3.1\u003csup\u003e50\u003c/sup\u003e, using lme4 package for all mixed models\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, MASS package\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e for using the quasi-binomial and quasi-poisson families in the GLMMs, and survival package for longevity data analyses\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. For all data, the significance level was set to \u0026alpha;\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge the Seychelles Bureau of Standards (SBS), the Ministry for Agriculture, Climate Change and Environment (MACCE), and the National Biosecurity Agency (NBA) for granting the necessary permits in Seychelles. We also extend our sincere thanks to the Seychelles Parks and Gardens Authority (SPGA), Club Med Sainte Anne, and the Moyenne Island Foundation for facilitating access to the Sainte Anne Marine National Park.\u003c/p\u003e\n\u003cp\u003eWe thank the Ministry of Health (MoH) and the University of Seychelles (UniSey) for their support throughout the project. This project was financially supported by the European Union, La R\u0026eacute;gion R\u0026eacute;union, and Le D\u0026eacute;partement de La R\u0026eacute;union. It was funded through two European Regional Development Fund (ERDF) Interreg programs: the SeyWol Project \u0026ndash; Phase I (INTERREG V OCEAN INDIEN 2014\u0026ndash;2020, No. 0031783) and the Operating SeyWol Project (INTERREG VI OCEAN INDIEN 2021\u0026ndash;2027, No. 004954), as well as by the Fonds de Coop\u0026eacute;ration R\u0026eacute;gionale 2021 (No. 001749). Finally, we warmly thank all individuals who contributed to the project, including Mr. David Labrosse from the Ministry of Health, and the staff at Jolly Roger Bar \u0026amp; Restaurant for their kind support.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.C. contributed to the study concept and design, conducted laboratory experiments and mass mosquito production, analyzed and interpreted the data, and wrote and prepared the manuscript and figures. B.G. contributed to the study design, analyzed and interpreted the data and was responsible for mass production of incompatible males and entomological surveys in Seychelles. M.D., D.S., A.J-B., and S.D. contributed to the mass production and release of incompatible males, as well as to entomological monitoring in Seychelles. M.A., H.D., H.P., K.S. and S.S. contributed to the entomological surveys. L.B. assisted in obtaining the necessary approvals to implement the field trial in Seychelles. J.F. and G.R. contributed to the logistical and technical administrative aspects. S.S., Q.L., and J.E. contributed to laboratory experiments, mass egg production in La R\u0026eacute;union, and molecular biology analyses. P.M. contributed to the initiation of the project in Seychelles. P.T. also contributed to the project\u0026rsquo;s initiation and to the preparation of the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary information The online version contains supplementary material available at\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePaupy, C., Delatte, H., Bagny, L., Corbel, V. \u0026amp; Fontenille, D. \u003cem\u003eAedes albopictus\u003c/em\u003e, an arbovirus vector: From the darkness to the light. \u003cem\u003eMicrobes Infect.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1177\u0026ndash;1185 (2009).\u003c/li\u003e\n\u003cli\u003eRezza, G. \u003cem\u003eAedes albopictus\u003c/em\u003e and the reemergence of Dengue. \u003cem\u003eBMC Public Health\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 72 (2012).\u003c/li\u003e\n\u003cli\u003eKraemer, M. U. G. \u003cem\u003eet al.\u003c/em\u003e Past and future spread of the arbovirus vectors \u003cem\u003eAedes aegypti\u003c/em\u003e and \u003cem\u003eAedes albopictus\u003c/em\u003e. \u003cem\u003eNat. Microbiol.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 854\u0026ndash;863 (2019).\u003c/li\u003e\n\u003cli\u003eAldridge, R. L., Gibson, S. \u0026amp; Linthicum, K. J. \u003cem\u003eAedes Aegypti\u003c/em\u003e controls \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003eAegypti\u003c/em\u003e: SIT and IIT-an overview. \u003cem\u003eJ. Am. Mosq. Control Assoc.\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 32\u0026ndash;49 (2024).\u003c/li\u003e\n\u003cli\u003eWerren, J. H. Biology of \u003cem\u003eWolbachia\u003c/em\u003e. \u003cem\u003eAnnu. Rev. Entomol.\u003c/em\u003e \u003cstrong\u003e124\u003c/strong\u003e, 587\u0026ndash;609 (1997).\u003c/li\u003e\n\u003cli\u003eZug, R. \u0026amp; Hammerstein, P. Still a host of hosts for \u003cem\u003eWolbachia\u003c/em\u003e: Analysis of recent data suggests that 40% of terrestrial arthropod species are infected. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 7\u0026ndash;9 (2012).\u003c/li\u003e\n\u003cli\u003eBeckmann, J. F., Ronau, J. A. \u0026amp; Hochstrasser, M. A \u003cem\u003eWolbachia\u003c/em\u003e deubiquitylating enzyme induces cytoplasmic incompatibility. \u003cem\u003eNat. Microbiol.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 17007 (2017).\u003c/li\u003e\n\u003cli\u003eLePage, D. P. \u003cem\u003eet al.\u003c/em\u003e Prophage WO genes recapitulate and enhance \u003cem\u003eWolbachia\u003c/em\u003e-induced cytoplasmic incompatibility. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e543\u003c/strong\u003e, 243\u0026ndash;247 (2017).\u003c/li\u003e\n\u003cli\u003eBoller, E. F., Russ, K., Vallo, V. \u0026amp; Bush, G. L. Incompatible races of European cherry fruit-fly, \u003cem\u003eRhagoletis cerasi\u003c/em\u003e (Diptera-Tephritidae), their origin and potential use in biological-Control. \u003cem\u003eEntomol. Exp. Appl.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 237\u0026ndash;247 (1976).\u003c/li\u003e\n\u003cli\u003eRiegler, M. \u0026amp; Stauffer, C. \u003cem\u003eWolbachia\u003c/em\u003e infections and superinfections in cytoplasmically incompatible populations of the European cherry fruit fly \u003cem\u003eRhagoletis cerasi\u003c/em\u003e (Diptera, Tephritidae). \u003cem\u003eMol. Ecol.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 2425\u0026ndash;2434 (2002).\u003c/li\u003e\n\u003cli\u003eCrawford, J. E. \u003cem\u003eet al.\u003c/em\u003e Efficient production of male \u003cem\u003eWolbachia\u003c/em\u003e-infected \u003cem\u003eAedes aegypti\u003c/em\u003e mosquitoes enables large-scale suppression of wild populations. \u003cem\u003eNat. Biotechnol.\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 482\u0026ndash;492 (2020).\u003c/li\u003e\n\u003cli\u003eBeebe, N. W. \u003cem\u003eet al.\u003c/em\u003e Releasing incompatible males drives strong suppression across populations of wild and \u003cem\u003eWolbachia\u003c/em\u003e-carrying \u003cem\u003eAedes aegypti\u003c/em\u003e in Australia. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, e2106828118 (2021).\u003c/li\u003e\n\u003cli\u003eConsortium, P. W.--S. \u0026amp; Ching, N. L. \u003cem\u003eWolbachia\u003c/em\u003e-mediated sterility suppresses \u003cem\u003eAedes aegypti\u003c/em\u003e populations in the urban tropics. \u003cem\u003eMedrxiv\u003c/em\u003e 2006\u0026ndash;2021 (2021).\u003c/li\u003e\n\u003cli\u003eRyff, K. R. \u003cem\u003eet al.\u003c/em\u003e Incompatible \u003cem\u003eAedes aegypti\u003c/em\u003e male releases as an intervention to reduce mosquito population \u0026mdash; A field trial in Puerto Rico. \u003cstrong\u003e19\u003c/strong\u003e, e 00128391 (2025) doi:10.1371/journal.pntd.0012839.\u003c/li\u003e\n\u003cli\u003eZeng, Q. \u003cem\u003eet al.\u003c/em\u003e A standalone incompatible insect technique enables mosquito suppression in the urban subtropics. \u003cem\u003eCommun. Biol.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 1\u0026ndash;13 (2022).\u003c/li\u003e\n\u003cli\u003eZheng, X. \u003cem\u003eet al.\u003c/em\u003e Incompatible and sterile insect techniques combined eliminate mosquitoes. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e572\u003c/strong\u003e, 56\u0026ndash;61 (2019).\u003c/li\u003e\n\u003cli\u003eBansal, S. \u003cem\u003eet al.\u003c/em\u003e Effectiveness of \u003cem\u003eWolbachia\u003c/em\u003e-mediated sterility coupled with sterile insect technique to suppress adult \u003cem\u003eAedes aegypti\u003c/em\u003e populations in Singapore: a synthetic control study. \u003cem\u003eLancet Planet. Heal.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e617\u0026ndash;e628 (2024).\u003c/li\u003e\n\u003cli\u003eChansang, C. \u003cem\u003eet al.\u003c/em\u003e Combined sterile insect technique and incompatible insect technique: The first proof-of-concept to suppress \u003cem\u003eAedes aegypti\u003c/em\u003e vector populations in semi-rural settings in Thailand. \u003cem\u003ePLoS Negl. Trop. Dis.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1\u0026ndash;21 (2019).\u003c/li\u003e\n\u003cli\u003eScussel, S. \u003cem\u003eet al.\u003c/em\u003e Combining transinfected \u003cem\u003eWolbachia\u003c/em\u003e and a genetic sexing strain to control \u003cem\u003eAedes albopictus\u003c/em\u003e in laboratory-controlled conditions. \u003cem\u003eProc. R. Soc. B\u003c/em\u003e \u003cstrong\u003e291\u003c/strong\u003e, 20240429 (2024).\u003c/li\u003e\n\u003cli\u003eCalvitti, M., Moretti, R., Lampazzi, E., Bellini, R. \u0026amp; Dobson, S. L. Characterization of a new \u003cem\u003eAedes albopictus\u003c/em\u003e (Diptera: Culicidae)\u0026ndash;\u003cem\u003eWolbachia pipientis\u003c/em\u003e (Rickettsiales: Rickettsiaceae) symbiotic association generated by artificial transfer of the \u003cem\u003ew\u003c/em\u003ePip strain from \u003cem\u003eCulex pipiens\u003c/em\u003e (Diptera: Culicida. \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 179\u0026ndash;187 (2010).\u003c/li\u003e\n\u003cli\u003eDobson, S. L., Fox, C. W. \u0026amp; Jiggins, F. M. The effect of \u003cem\u003eWolbachia\u003c/em\u003e-induced cytoplasmic incompatibility on host population size in natural and manipulated systems. \u003cem\u003eProc. R. Soc. B Biol. Sci.\u003c/em\u003e \u003cstrong\u003e269\u003c/strong\u003e, 437\u0026ndash;445 (2002).\u003c/li\u003e\n\u003cli\u003eHoffmann, A. A. \u0026amp; Michael, T. Cytoplasmic incompatibility in insects. \u003cem\u003eInflu. passengers\u003c/em\u003e 42\u0026ndash;80 (1997).\u003c/li\u003e\n\u003cli\u003eMoretti, R., Marzo, G. A., Lampazzi, E. \u0026amp; Calvitti, M. Cytoplasmic incompatibility management to support Incompatible Insect Technique against \u003cem\u003eAedes albopictus\u003c/em\u003e. \u003cem\u003eParasites and Vectors\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, (2018).\u003c/li\u003e\n\u003cli\u003eLi, Y. \u003cem\u003eet al.\u003c/em\u003e Reply to: Issues with combining incompatible and sterile insect techniques. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e590\u003c/strong\u003e, E3-E5 (2021).\u003c/li\u003e\n\u003cli\u003eMoretti, R. \u0026amp; Calvitti, M. Issues with combining incompatible and sterile insect techniques. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e590\u003c/strong\u003e, E1\u0026ndash;E2 (2021).\u003c/li\u003e\n\u003cli\u003eDenton, J. A., Joubert, D. A., Goundar, A. A. \u0026amp; Gilles, J. R. L. International shipments of \u003cem\u003eWolbachia\u003c/em\u003e infected mosquito eggs: towards the scaling-up of World Mosquito Program operations. \u003cem\u003eOIE Rev. Sci. Tech.\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 91\u0026ndash;99 (2022).\u003c/li\u003e\n\u003cli\u003eDobson, K. L. \u003cem\u003eet al.\u003c/em\u003e Satellite rearing of \u003cem\u003eAedes\u003c/em\u003e mosquito eggs: synchronized empirical test of a novel mass rearing model. \u003cem\u003eJ. Am. Mosq. Control Assoc.\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 12\u0026ndash;17 (2023).\u003c/li\u003e\n\u003cli\u003eLejarre, Q. \u003cem\u003eet al.\u003c/em\u003e Development of the Incompatible Insect Technique targeting \u003cem\u003eAedes albopictus\u003c/em\u003e: introgression of a wild nuclear background restores the performance of males artificially infected with \u003cem\u003eWolbachia\u003c/em\u003e. \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e e02350-24 (2025).\u003c/li\u003e\n\u003cli\u003eGoubert, C., Minard, G., Vieira, C. \u0026amp; Boulesteix, M. Population genetics of the Asian tiger mosquito \u003cem\u003eAedes albopictus\u003c/em\u003e , an invasive vector of human diseases. \u003cem\u003eHeredity (Edinb).\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 125\u0026ndash;134 (2016).\u003c/li\u003e\n\u003cli\u003eRezza, G. \u003cem\u003eet al.\u003c/em\u003e Infection with chikungunya virus in Italy: an outbreak in a temperate region. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e370\u003c/strong\u003e, 1840\u0026ndash;1846 (2007). \u003c/li\u003e\n\u003cli\u003eFrumence, E. \u003cem\u003eet al.\u003c/em\u003e Genomic insights into the re-emergence of chikungunya virus on R\u0026eacute;union Island, France, 2024 to 2025. \u003cem\u003eEurosurveillance\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1\u0026ndash;6 (2025).\u003c/li\u003e\n\u003cli\u003eSant\u0026eacute; publique France (SpF). Bilan de la surveillance renforc\u0026eacute;e de la dengue , du chikungunya et du Zika en France hexagonale du 1 er mai au 17 juin 2025 Chikungunya , dengue et Zika Episodes de transmission autochtone. 1\u0026ndash;8 (2025). French. Available from: https://www.santepubliquefrance.fr/maladies-et-traumatismes/maladies-a-transmission-vectorielle/chikungunya/documents/bulletin-national/chikungunya-dengue-et-zika-en-france-hexagonale.-bulletin-de-la-surveillance-renforcee-du-17-juin-2025\u003c/li\u003e\n\u003cli\u003eGuo, J., Zheng, X., Zhang, D. \u0026amp; Wu, Y. Current Status of Mosquito Handling, Transporting and Releasing in Frame of the Sterile Insect Technique. \u003cem\u003eInsects\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 532 (2022).\u003c/li\u003e\n\u003cli\u003eBenedict, M. Q. Sterile Insect Technique: Lessons from the Past. \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 1974\u0026ndash;1979 (2021).\u003c/li\u003e\n\u003cli\u003eGarcia, G. de A. \u003cem\u003eet al.\u003c/em\u003e Matching the genetics of released and local \u003cem\u003eAedes aegypti\u003c/em\u003e populations is critical to assure \u003cem\u003eWolbachia\u003c/em\u003e invasion. \u003cem\u003ePLoS Negl. Trop. Dis.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1\u0026ndash;23 (2019).\u003c/li\u003e\n\u003cli\u003eRoss, P. A. Designing effective \u003cem\u003eWolbachia\u003c/em\u003e release programs for mosquito and arbovirus control. \u003cem\u003eActa Trop.\u003c/em\u003e \u003cstrong\u003e222\u003c/strong\u003e, 106045 (2021).\u003c/li\u003e\n\u003cli\u003eBeebe, N. W. \u003cem\u003eet al.\u003c/em\u003e Releasing incompatible males drives strong suppression across populations of wild and \u003cem\u003eWolbachia\u003c/em\u003e-carrying \u003cem\u003eAedes aegypti\u003c/em\u003e in Australia. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eGong, J.-T. \u003cem\u003eet al.\u003c/em\u003e Upscaling the production of sterile male mosquitoes with an automated pupa sex sorter. \u003cem\u003eSci. Robot.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, eadj6261 (2024).\u003c/li\u003e\n\u003cli\u003eAtyame, C. M., Delsuc, F., Pasteur, N., Weill, M. \u0026amp; Duron, O. Diversification of \u003cem\u003eWolbachia\u003c/em\u003e endosymbiont in the \u003cem\u003eCulex pipiens\u003c/em\u003e mosquito. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 2761\u0026ndash;2772 (2011).\u003c/li\u003e\n\u003cli\u003eLebon, C., Benlali, A., Atyame, C., Mavingui, P. \u0026amp; Tortosa, P. Construction of a genetic sexing strain for \u003cem\u003eAedes albopictus\u003c/em\u003e: A promising tool for the development of sterilizing insect control strategies targeting the tiger mosquito. \u003cem\u003eParasites and Vectors\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, (2018).\u003c/li\u003e\n\u003cli\u003ePoinsot, D., Bourtzis, K., Markakis, G., Savakis, C. \u0026amp; Mer\u0026ccedil;ot, H. \u003cem\u003eWolbachia\u003c/em\u003e \u003cem\u003eT\u003c/em\u003eransfer from \u003cem\u003eDrosophila melanogaster\u003c/em\u003e into \u003cem\u003eD\u003c/em\u003e. \u003cem\u003esimulans\u003c/em\u003e: \u003cem\u003eH\u003c/em\u003eost \u003cem\u003eE\u003c/em\u003effect and \u003cem\u003eC\u003c/em\u003eytoplasmic \u003cem\u003eI\u003c/em\u003encompatibility \u003cem\u003eR\u003c/em\u003eelationships. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e150\u003c/strong\u003e, 227\u0026ndash;237 (1998).\u003c/li\u003e\n\u003cli\u003eCattel, J. \u003cem\u003eet al.\u003c/em\u003e Back and forth \u003cem\u003eWolbachia\u003c/em\u003e transfers reveal efficient strains to control spotted wing drosophila populations. \u003cem\u003eJ. Appl. Ecol.\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 2408\u0026ndash;2418 (2018).\u003c/li\u003e\n\u003cli\u003eVavassori, L., Saddler, A. \u0026amp; M\u0026uuml;ller, P. Active dispersal of \u003cem\u003eAedes albopictus\u003c/em\u003e: A mark-release-recapture study using self-marking units. \u003cem\u003eParasites and Vectors\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1\u0026ndash;14 (2019).\u003c/li\u003e\n\u003cli\u003eSeber, G. A. F. \u003cem\u003eThe estimation of animal abundance and related parameters\u003c/em\u003e. (1982).\u003c/li\u003e\n\u003cli\u003ePollock, K. H., Nichols, J. D., Brownie, C. \u0026amp; Hines, J. E. Statistical inference for capture-recapture experiments. \u003cem\u003eWildl. Monogr.\u003c/em\u003e 3\u0026ndash;97 (1990).\u003c/li\u003e\n\u003cli\u003eCianci, D. \u003cem\u003eet al.\u003c/em\u003e Estimating mosquito population size from mark-release-recapture data. \u003cem\u003eJ. Med. Entomol.\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 533\u0026ndash;542 (2013).\u003c/li\u003e\n\u003cli\u003eAtyame, C. M. \u003cem\u003eet al.\u003c/em\u003e Comparison of irradiation and \u003cem\u003eWolbachia\u003c/em\u003e based approaches for sterile-male strategies targeting \u003cem\u003eAedes albopictus\u003c/em\u003e. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1\u0026ndash;15 (2016).\u003c/li\u003e\n\u003cli\u003eCollins, F. \u003cem\u003eet al.\u003c/em\u003e Construction of a general human chromosome jumping library, with application to cystic fibrosis. \u003cem\u003eScience \u003c/em\u003e\u003cstrong\u003e235\u003c/strong\u003e, 1046\u0026ndash;1050 (1987).\u003c/li\u003e\n\u003cli\u003eDuron, O. \u003cem\u003eet al.\u003c/em\u003e Variability and expression of ankyrin domain genes in \u003cem\u003eWolbachia\u003c/em\u003e variants infecting the mosquito \u003cem\u003eCulex pipiens\u003c/em\u003e. \u003cem\u003eJ. Bacteriol.\u003c/em\u003e \u003cstrong\u003e189\u003c/strong\u003e, 4442\u0026ndash;4448 (2007).\u003c/li\u003e\n\u003cli\u003eR Core Team. R: A Language and Environment for Statistical Computing. (2023).\u003c/li\u003e\n\u003cli\u003eBates, D. \u003cem\u003eet al.\u003c/em\u003e Package \u0026lsquo;lme4\u0026rsquo;. \u003cem\u003eConvergence\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 2 (2015).\u003c/li\u003e\n\u003cli\u003eRipley, B. \u003cem\u003eet al.\u003c/em\u003e Package \u0026lsquo;mass\u0026rsquo;. \u003cem\u003eCran r\u003c/em\u003e \u003cstrong\u003e538\u003c/strong\u003e, 113\u0026ndash;120 (2013).\u003c/li\u003e\n\u003cli\u003eTherneau, T. M. A Package for Survival Analysis in R. (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6936662/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6936662/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eClimate and land-use changes are accelerating the spread of the mosquito \u003cem\u003eAedes albopictus\u003c/em\u003e, a major arbovirus vector, leading to the emergence and autochthonous transmission of Dengue or Chikungunya viruses in temperate regions such as Italy and France. This situation is stimulating the development of innovative vector control strategies allowing to overcome the rapid selection of insecticide resistance. The Incompatible Insect Technique (IIT) allows suppressing mosquito populations through inundative releases of artificially \u003cem\u003eWolbachia\u003c/em\u003e infected males that sterilize local females through Cytoplasmic Incompatibility (CI). We carried out a six-month IIT suppression trial on a remote island located in the Western Indian Ocean. We used a recently constructed and optimized \u003cem\u003eAedes albopictus\u003c/em\u003e transinfected line sheltering a single \u003cem\u003eWolbachia\u003c/em\u003e infection and inducing bi-directional CI. This feature ensures that released males sterilize local females, while infected females resulting from accidental releases are also sterilized by wild-type males, thereby preventing population replacement, a key limitation of conventional IIT. The trial was conducted in operational conditions: mosquito populations were monitored during suppression and the number of released males was adjusted based on wild population density. Importantly, eggs were produced in a central insectary located over 1,000 km from the release area, transported via commercial flights to a satellite insectary for male production, and finally shipped by boat to the release site. Our results demonstrated that (i) over 95% suppression can be achieved within a few weeks of treatment, (ii) as expected the use of a mono-infected line prevented population replacement, (iii) large-scale shipment of eggs under operational conditions is both feasible and effective, supporting the scalability and industrial deployment of this environmental-friendly vector control strategy.\u003c/p\u003e","manuscriptTitle":"Production and shipment of Wolbachia-infected eggs allow controlling Aedes albopictus through the Incompatible Insect Technique on a remote island","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-21 13:44:03","doi":"10.21203/rs.3.rs-6936662/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c81e4f8c-deef-42a1-b667-2f2c027c1d73","owner":[],"postedDate":"July 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50811290,"name":"Biological sciences/Biotechnology/Environmental biotechnology"},{"id":50811291,"name":"Biological sciences/Microbiology/Applied microbiology"}],"tags":[],"updatedAt":"2025-12-30T08:10:53+00:00","versionOfRecord":{"articleIdentity":"rs-6936662","link":"https://doi.org/10.1038/s42003-025-09269-0","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-11-26 05:00:00","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-07-21 13:44:03","video":"","vorDoi":"10.1038/s42003-025-09269-0","vorDoiUrl":"https://doi.org/10.1038/s42003-025-09269-0","workflowStages":[]},"version":"v1","identity":"rs-6936662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6936662","identity":"rs-6936662","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}