# 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, Quentin Lejarre, Jeremy Esnault, Daryl Sinon, Alvin Jean-Bonnelame, Stéphanie Donet, Merna Amade, et al.

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## To cite this version:

Julien Cattel, Benjamin Gaudillat, Marianne Duployer, Sarah Scussel, Quentin Lejarre, et al. Production and shipment of Wolbachia-infected eggs allow controlling Aedes albopictus through the Incompatible Insect Technique on a remote island. 2025. hal-05389951

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Distributed under a Creative Commons Attribution 4.0 International License
# Production and shipment of Wolbachia-infected eggs allow controlling Aedes albopictus through the Incompatible Insect Technique on a remote island

Julien Cattel

juliencattel@gmail.com

SAS SYMBIOTIC https://orcid.org/0000-0002-8715-586X

Benjamin Gaudillat

Groupement d'intérêt Public Cyclotron Océan Indien (CYROI)

Marianne Duployer

Groupement d'intérêt Public Cyclotron Océan Indien (CYROI)

Sarah Scussel

Groupement d'intérêt Public Cyclotron Océan Indien (CYROI)

Quentin Lejarre

SAS SYMBIOTIC

Jeremy Esnault

Groupement d'intérêt Public Cyclotron Océan Indien (CYROI)

Daryl Sinon

Island Biodiversity and Conservation center (IBC)

Alvin Jean-Bonnelame

Island Biodiversity and Conservation center (IBC)

Stéphanie Donet

Island Biodiversity and Conservation center (IBC)

Merna Amade

Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health

Hilary Desir

Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health

Hamid Pool

Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health

Kenneth Sinon

Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health

Steeve Savy

Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health

Nigel Sultan
Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health

**Kérina Jean-Baptiste**

Vector Control Unit, Public Health Services, Public Health Authority, Ministry of Health

**Leon Biscornet**

Seychelles Public Health Laboratory, Public Health Authority, Ministry of Health

https://orcid.org/0000-0001-6074-6582

**Joseph François**

Island Biodiversity and Conservation center (IBC)

**Gerard Rocamora**

Island Biodiversity and Conservation Centre, University of Seychelles

**Patrick MAVINGUI**

UMR PIMIT (Processus Infectieux en Milieu Insulaire Tropical)

**Pablo Tortosa**

Université de La Réunion, Unité Mixte de Recherche Processus Infectieux en Milieu Insulaire Tropical
(UMR PIMIT), CNRS 9192, INSERM 1187, IRD 249

**Article**

**Keywords:**

**Posted Date:** July 21st, 2025

**DOI:** https://doi.org/10.21203/rs.3.rs-6936662/v1

**License:** © Ⓟ This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** There is **NO** Competing Interest.
**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.

**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 arboviruses¹. 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 virus²,³. 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 populations⁴.
IIT leverages the properties of *Wolbachia pipientis*, a maternally inherited endosymbiotic bacterium that infects approximately 40% of arthropod species⁵,⁶. *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* strain⁵. 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 sperm⁷,⁸.

CI can be hijacked for the development of the IIT, which involves the release of *Wolbachia*-infected males that are incompatible with resident females⁹,¹⁰. Recent studies have demonstrated the effectiveness of standalone IIT in controlling *Ae. aegypti* populations in the U.S¹¹, Australia¹², Singapore¹³, and Puerto Rico¹⁴ as well as *Ae. albopictus* populations in China¹⁵. 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 eliminate¹⁶⁻¹⁸. 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 CI¹⁹,²⁰. 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 invasion²², 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 program⁴. 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 term²³. 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 terms²⁴,²⁵, 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<sup>26,27</sup>. 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* wPip transinfected line (Scussel et al., 2024) introgressed with a wild genetic background from the Seychelles, resulting in the SEY-wPip line. Reciprocal crosses between wild lines and SEY-wPip confirmed that wPip induced bidirectional CI in *Ae. albopictus* regardless of the host genetic background<sup>19,28</sup>. Indeed, when SEY-wPip 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-wPip females, the hatching rate ranged from 0.47 to 9.41% (Table 1).

<table><caption>Table 1
Hatch rate obtained in control and reciprocal crosses between the SEY-wPip and three different wild lines.</caption><thead><tr><th rowspan="2">crosses</th><th>nb of hatched</th><th>nb of unhatched eggs</th><th>hatch rate (%) (± SD)</th><th>CI (%)</th><th>Clcorr (%)</th></tr><tr><th>eggs</th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>¤ SEY-wPip x ¤ Le Port</td><td>78</td><td>9,053</td><td>1,05 (± 1,67)</td><td>98,95</td><td>92,93</td></tr><tr><td>¤ SEY-wPip x ¤ S-RUN</td><td>45</td><td>8,007</td><td>0,59 (± 0,70)</td><td>99,41</td><td>97,38</td></tr><tr><td>¤ SEY-wPip x ¤ La Providence</td><td>17</td><td>4,683</td><td>0,37 (± 0,07)</td><td>99,63</td><td>98,33</td></tr><tr><td>¤ Le Port x ¤ SEY-wPip</td><td>61</td><td>7,491</td><td>0,76 (± 0,31)</td><td>99,24</td><td>96,34</td></tr><tr><td>¤ S-RUN x ¤ SEY-wPip</td><td>25</td><td>5,401</td><td>0,47 (± 0,21)</td><td>99,53</td><td>97,74</td></tr><tr><td>¤ La Providence x ¤ SEY-wPip</td><td>465</td><td>4,562</td><td>9,41 (± 2,96)</td><td>90,59</td><td>54,69</td></tr><tr><td>¤ Le Port x ¤ Le Port</td><td>10,468</td><td>1,701</td><td>85,14 (± 7,30)</td><td></td><td></td></tr><tr><td>¤ S-RUN x ¤ S-RUN</td><td>5,416</td><td>1,652</td><td>77,51 (± 4,92)</td><td></td><td></td></tr><tr><td>¤ La Providence x ¤ La Providence</td><td>1,918</td><td>563</td><td>77,88 (± 5,80)</td><td></td><td></td></tr><tr><td>¤ SEY-wPip x ¤ SEY-wPip</td><td>9,875</td><td>2,483</td><td>79,23 (± 7,94)</td><td></td><td></td></tr></tbody></table>

## Life history traits (LHTs) and mating competitiveness of the SEY- w Pip line in laboratory conditions
To assess the fitness of the *SEY-*wPip 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-*wPip lines (log-rank test; *P* < 0.001 for both comparisons), and *SEY-*wPip also had a longer longevity than S-RUN, although this difference was significant in males only (Supp. Figure 1A-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-*wPip 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 1C). 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-*wPip line showing a mean value of 75.89% (± 25.75) (Supp. Figure 1D).

Finally, we assessed the mating competitiveness of *SEY-*wPip 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-*wPip) 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 1E). Of note, when different combinations of *SEY-*wPip 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-*wPip 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-*wPip appears as a mono-transinfected line suitable for mass-production and subsequent use in an IIT program, as the potential fitness cost associated with *wPip* 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 2A), 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 2B).

Three months before the start of incompatible male releases, eggs of the SEY-*wPip* 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. 2A). 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-*wPip* 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. 2B). 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. 2C). 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-*wPip* 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-*wPip* to wild males). After twelve weeks of releases, this resulted in an overall population suppression level of 95% (Fig. 2C).

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. 2A). 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-*wPip* 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. 2B-C), with a male release ratio remaining at
1:1 (SEY-*wPip* 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-Cl 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. 4A). 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 *wPip* females at the study site (see below).

Thus, to monitor the risk of SEY-*wPip* 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 *wPip*-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 *wPip*-positive rate ranging from 0 to 6.3% during the release period, with a maximum of three positive ovitraps (Fig. 4B). Although *wPip*-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<sup>29-32</sup>. 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<sup>33</sup>. 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-Cl 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-*wPip 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-*wPip 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<sup>28,34-36</sup>.

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-*wPip* 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 program¹⁵. 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 *wPip*-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 rate¹¹,¹⁴,³⁷. 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 unnecessary²⁷. 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 *wPip-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 F₁ post sampling) with 4–5 day-old virgin females from the transinfected line for four consecutive generations, resulting in a line named *SEY-*wPip, 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-*wPip 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 Island²⁸,⁴⁰, 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-*wPip 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 expected¹⁹, 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:
$CI_{corr} = [(CI_{obs} - CCM)/(100 - CCM)] \times 100$, 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-*wPip* line were compared to those of the S-RUN and Le Port lines, both showing high fitness under laboratory-controlled conditions¹⁹,²⁸. 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-*wPip* 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-*wPip*), 1 : 5 (17% S-RUN or Le Port : 85% SEY-*wPip*), 1 : 10 (9% S-RUN or Le Port : 90% SEY-*wPip*) and two control ratios, 1 : 0 (100% S-RUN or Le Port: 0% SEY-*wPip*) and 0 : 1 (0% S-RUN or Le Port: 100 SEY-*wPip*). 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 prototype⁴³. 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, m the number of captured mosquitos, M the number of released marked mosquitos, and m the number of recaptured marked mosquitos<sup>44-46</sup>.

**Mass Production and release of incompatible males**

Mass production and subsequent release of SEY-*wPip* 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. 2A). 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 described⁴⁷.

## 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, respectively¹⁵:

$$SE (\%) = \frac{Hc - Hr}{Hc} \times 100$$

## 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 *wPip*-positive adults were monitored at eleven different time points, with a total of 1,196 individuals checked for the presence or absence of *wPip*. Two indicators were used to monitor the risk: (i) the proportion of ovitraps containing *wPip*-positive adults and (ii) the proportion of *wPip*-positive individuals emerging from each positive ovitrap.

## DNA extraction and *wPip* screening

Genomic DNA was extracted from adult mosquitoes using the cetyltrimethylammonium bromide (CTAB) method⁴⁸. 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-TCCATATCGATCTACTGCGT⁴⁹. 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/g/mmFAQ.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 4.3.1⁵⁰, using lme4 package for all mixed models⁵¹, MASS package⁵² for using the quasi-binomial and quasi-poisson families in the GLMMs, and survival package for longevity data analyses⁵³. 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

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# Figures

<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>

Control and release sites, located within the Sainte Anne Marine National Park in the Seychelles. The control site (7 ha) is a part of Sainte-Anne island, while the release site corresponds to the entire surface of Moyenne island.
<figure><img src="image_4.png" /><figcaption>**Figure 2**</figcaption></figure>

**Suppression of the wild population.** A Total number of SEY-*wPip* males released each week during the release period. B Average hatching rate per week in the release and control sites. C 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.
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>

**Spatial-temporal dynamics of hatched eggs at the release and control sites for each release phase.** The different color indicates the average number of hatched eggs.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>Figure 4</figcaption></figure>

**Temporal dynamics of *wPip* females in laboratory male production and at the release site.** A Female contamination rates measured in SEY-*wPip* 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. B *wPip*-positive individual rate detected at the release site measured using ovitraps. For each
screening we indicated the number of *wPip-positive* individuals and the total number of screened adults.
Between brackets: total number of ovitraps for which at least one *wPip-positive* individual was detected.

**Supplementary Files**

This is a list of supplementary files associated with this preprint. Click to download.

* dataLHTfecundityhatchratelabSuppFig1.csv

* dataClcrosses.xlsx

* dataLHTmalecompetitivenesslabSuppFig1.csv

* datawPipfemalecontaminationreleasesiteFig4.xlsx

* datanumberhatchedeggshatchrateFig2.csv

* dataMRR.xlsx

* dataLHTlongevitylabSuppFig1.csv

* SupplementaryFigures.docx