Integrated Control of Aedes Albopictus (Diptera: Culicidae) in Southwest Germany supported by the Sterile Insect Technique

The invasive species Aedes albopictus, the Asian tiger mosquito, has undergone an extreme expansion by steady introductions as blind passengers in vehicles from the Mediterranean to South-West Germany. The more than 15 established populations in the State of Baden-Württemberg and Palatine (South-West Germany) have become a major nuisance and public health threat. Aedes albopictus deserves special attention as vector of arboviruses like dengue, chikungunya or Zika virus. In Germany, control of Ae. albopictus is implemented under the auspice of health departments and regulatory oces. Methods The comprised or a) based on the or of breeding sites with the use of (Culinex plus); b) Door-to-Door (DtD) control by trained staff applying high doses of a Bti-water-dispersible granular formulation (Vectobac WG) aimed for a long-lasting killing effect; and c) the Sterile Insect Technique (SIT) to eliminate remaining Ae. albopictus populations. Prior to large scale routine city-wide treatments, the ecacy of the three elements was evaluated in laboratory and semi-eld trials. Special emphasis was given to the mass release of Ae. albopictus sterile males.

remaining Ae. albopictus population to a minimum or even to eradicate it. The combination of the use of Bti with SIT are most effective and selective tools against Ae. albopictus, one of the most dangerous mosquito vector species.

Background
Among the more than 3,500 known mosquito species, about 30 have spread beyond their original geographical borders [1,2]. Several invasive species have a severe impact on public health, not only in the tropics but also in temperate climates. Aedes aegypti, Ae. albopictus, Ae. japonicus, Ae. koreicus, Ae. atropalpus and Ae. triseriatus belong to this group.
Aedes albopictus deserves special attention as a vector of at least 22 arboviruses, including dengue, chikungunya, Zika and yellow fever viruses [3][4][5][6][7][8]. Since 1990, Ae. albopictus took a permanent foothold in Europe. It had been introduced to Italy by the international trade of used tires. From Genoa [9,10], Ae. albopictus was spread as a blind passenger via road, rail and boats over Italy and further along the Mediterranean coast to France, Spain, the Balkan, Greece and Turkey [57]. Today, the species is rmly established in the whole European Mediterranean basin. It has started spreading across the Alps into central Europe. Autochthonous transmissions vectored by Ae. albopictus of chikungunya, dengue and Zika viruses have ared up in the Mediterranean region [6,7,8].
Realizing the risk of passive transport of Ae. albopictus from Italy to Germany by road and rail, especially during the summer holiday season, the German Mosquito Control Association (KABS) started a monitoring program in 2005 [2,19]. Surveillance of resting stations and camping grounds along the highway A5 leading from Italy revealed during the summer months a regular and increasing appearance of Ae. albopictus along the Upper Rhine area [2]. A rst established population was reported in an allotment garden in Freiburg in September 2014. By 2019 at least twenty cities along the upper Rhine valley were infested by overwintering populations of Ae. albopictus ( Fig. 1) [55]. In all colonised areas, immediate surveillance was initiated by means of ovitraps, larval sampling and human bait collections to assess the size of the infested area and the abundance of Ae. albopictus. The goal was to detect and eliminate the breeding sites and to start eradication programmes.
After an in-depth evaluation of control tools in a previous study [24], it was decided to base the control strategy on three pillars: 1) community participation (CP) including distribution of Bti-zzy-tablets containing the protoxins of Bacillus thuringiensis israelensis (Bti), 2) Door to Door (DtD) activities including the removal or treatment of all breeding sites with a Bti-suspension by trained staff with Bti at three weeks intervals, and 3) the integration of the Sterile Insect Technique (SIT), an environmentally-friendly insect pest control method, to wipe out remaining Ae. albopictus populations originated from cryptic and/or non-accessible breeding sites.

Community participation (CP)
Community participation (CP) focuses on the increase in public awareness to prevent mosquito breeding and to record the occurrence of Ae. albopictus as an "early warning system". The CP includes detailed information provided to the public via press releases, TV air time, yers, web pages and information events, e.g. at schools, in city halls or meetings of gardener associations. Thus, public awareness was strengthened by detailed information on the characteristics, distribution, and the biology of the Asian tiger mosquito. In addition, measures are communicated to prevent the proliferation of the mosquito. This included the elimination of breeding sites, environmental sanitation, e.g. by the use of rmly tting lids to water containers and the treatment with Bti tablets (Culinex Tab plus). Bti tablets are distributed to the public in support of the DtD activities conducted by the expert teams. Additionally, in heavily infested areas, mosquito nets were distributed free of charge to thoroughly cover rainwater containers preventing access to female mosquitoes thus the proliferation of vectors.

Door-to-Door (DtD) control and Bti-treatments
Due to the lack of professional know-how and active involvement CP alone was not enough to reach the goal of strongly reducing or even eliminating the Asian tiger mosquito [59]. Therefore, DtD control of Ae. albopictus by trained staff was implemented. This measure along with long-lasting Bti-treatments was highly effective. Ae. albopictus populations could be strongly reduced or even eliminated.

The Sterile Insect Technique
The nal goal was a signi cant reduction or ideally the elimination of Ae. albopictus populations. Therefore, the Sterile Insect Technique (SIT) was added as third pillar to the integrated control strategy using gammairradiated sterile males. Ae. albopictus is particularly suitable for employing SIT, as the species is easily massreared, has a limited ight range, does not reproduce in enormous masses within a very short period like oodwater mosquitoes, and breeding sites are well de ned in urban areas mainly [29].
Therefore, the SIT method is an excellent tool to access also those breeding sites which are out of reach of standard control measures. This applies also to property owners refusing entry permission.
Preceding the release of sterile males, the natural Ae. albopictus population has to be low or strongly reduced by CP and DtD control. The sterile males have to outcompete their wild counterparts resulting in a large majority of wild females laying sterile eggs [30].

Final goals of the three-pillar approach
The goal of the pilot program was to assess the effect of the three-pillar control strategy against Ae. albopictus starting in the laboratory, extending to semi-eld tests and ending in routine eld applications. Ovitraps served as the main monitoring tool. A cost-analysis is presented to serve as a guideline for further planning of community-based control activities.

Study areas
The large-scale study was conducted in three Ae. albopictus infested large areas in Southern Germany: a) The "Melm" district (65 ha) within the City of Ludwigshafen (Palatine). This is a well-de ned residential area, developed at the end of the last century. It consists of about 950 family homes with gardens and some apartment buildings. Abundant breeding sites of Ae. albopictus were present. The 65 ha were subdivided into three sectors of almost equal size, each with speci c mosquito control scheme: Sector A: 23 ha (CP + DtD); Sector B (CP + DtD + SIT): 17 ha; and Sector C: 25 ha (CP + DtD) (Fig. 2).
b) the Metzgergrün area within the city of Freiburg (Baden-Württemberg). The selected site has a size of 4.5 ha comprising mostly apartment buildings for social housing (Fig. 3). It contains in adjacent gardens a large number of potential breeding sites such as used tires and water catching garbage. In this area, the SIT technique was employed in addition to CP and DtD.
c) Gartenstadt (Freiburg): This site served as control area (4 ha) and is about 1 km away from Metzgergrün.
The infestation by Ae. albopictus was comparable to the Metzgergrün.

Design and layout
The large-scale eld study was based on a three-pillar strategy, namely community participation (CP), door-todoor control (DtD), and sterile insect technique (SIT).

Community participation
In the rst week of May 2020, before the program started, citizens in Melm were informed via local media about the planned control activities against Tiger mosquitoes. The goal was to turn the residents from spectators to actors in the ght against Ae. albopictus. Between the 9th and 12th of May, three people distributed yers to 1820 households with detailed information helping the residents to control the Asian tiger mosquito on their properties. Each household received a yer with instructions and attached to each yer was a card board box containing 10 Bti-tablets in a blister pack (Culinex® Tab plus, Lot: 0604783, activity: 1000 ITU/mg, Culinex GmbH, Ludwigshafen).
During the DtD activities, the number of accessed and non-accessed properties as well as the presence of container-breeding mosquitoes were recorded to determine the e cacy of CP. Furthermore, the residents were interviewed regarding their knowledge on mosquito control. During the last control round (no. 5) in September, all available residents were asked if they had implemented the proposed control activities on their property.

DtD control
The most powerful tool to control Ae. albopictus was the DtD approach by trained staff applying long-lasting Bti-treatments. Therefore, in the rst step it was crucial to assess the optimum dosage for the Bti-treatments in large and small water collections.

Assessment of the optimum effective dosage for Btitreatments
Whereas the control of oodwater mosquitoes requires only a single effective dosage of Vectobac WG (strain AM5265, Valent Biosciences, Libertyville, USA) [56], the control of container-breeding species needs a long-term residual activity due to the constant follow-up of generations. Thus, the sequence of retreatments and the costs for manpower could be strongly reduced when a long-term effect of several weeks (at least 3 weeks) is achieved.
In a rst series of tests, the effect of Bti-treatments in rainwater containers was simulated. The Bti mixture was prepared with Vectobac WG with an activity 2700 ITUs/mg by thoroughly mixing 250 g of Vectobac WG with 1.5 l of tap water (pH: 7.8; conductivity: 680 µS; 0.675x10 9 ITU/1500 ml). Aliquots of the suspension were taken with an Eppendorf pipette by continuous stirring and applied to plastic buckets lled with 20 litres of tap water (1/10 of the usual water volume of a regular rainwater container). From the stock suspension 6 ml, 0.6 ml and 0.06 respectively were added to the 20 l of container water. This corresponded to 6 g, 0.6 g and 0.06 g of Vectobac-WG. Each dosage was tested in four replicates at a constant temperature of 24°C ± 1°C, with 4 buckets serving as a control. Twenty third instar larvae of laboratory-reared Ae. albopictus were added to each bucket. The mortality reading was taken at 24 and 48h post-treatment. At weekly intervals, 5 l of water was removed and replaced with the same amount tap water to simulate natural conditions when water is removed for watering the garden. Then, 20 third instar larvae were again added to each container. At each 48h reading larval cadavers were removed. The experiment was run until the mortality rates fell below 60%.
In a second series, the e cacy of Vectobac WG was tested in four different small water containers typically present in garden areas: a) terracotta owerpots with rough surfaces (volume: 1,400 ml); b) terracotta pots with smooth walls (volume: 1,400 ml); c) plastic owerpots (volume: 950 ml); d) zinc pots (volume: 800 ml); and e) terracotta owerpot saucers (volume: 200 ml). Before application, the recipients were scrubbed with ower soil (Compo Sana potting soil), cleaned with water and dried for 24 h to simulate natural conditions. The inside of four empty containers of each type were homogenously sprayed with a Mesto pressurised sprayer (Mesto BUGSI 1.5L), with 15 ml of Vectobac WG stock solution (250 g Vectobac WG mixed with 1.5 L of tap water; 0.675x 109 ITUs/1500 ml) resulting in 2.5 g Vectobac WG/small container (6.75x10 6 ITUs/container). The containers were dried for 48 hours and lled with tap water. Twenty Ae. albopictus third instar larvae were added to each container. The mortality reading was done at 48 h post-treatment. The containers were emptied after the mortality reading and dried again for 48 hours, re lled with water and stocked with a new batch of larvae. The procedure was repeated until the mortality rates fell below 60%. Four containers of each type served as untreated control. The test was conducted at 24°C ± 1.5°C and 80% RH.

Routine Bti-treatments
The eld staff consisted of six persons with in-depth knowledge on mosquito biology and taxonomy, especially breeding habits of Ae. albopictus. The staff had to handle the application of Bti at predetermined dosages to each potential breeding site as well as evaluate the mosquito traps at regular intervals for surveillance purposes. All accessible properties were inspected from June to October at intervals of three weeks. All potential breeding sites were treated with Vectobac WG. Finally, keeping close contact with the residents of nearly 2000 households was essential, providing information and Bti-tablets. Between May and September, the program included ve rounds of surveillance, control and data collection. The data were entered into a geographic information system (GIS).
Melm in Ludwigshafen: Biting Ae. albopictus were signalled for the rst time in early August 2019. The county health department and the city authorities were informed. Ae. albopictus populations were found along four streets. In addition, scattered across the district, more breeding sites (unused owerpots, saucers, rainwater barrels) were identi ed. Gullies were not functioning as breeding sites because all the water collected, runs off directly into the sewage plants. In total, 55 potential breeding sites were inspected with six positives for Ae. albopictus (CI: 10.9%). Thus, in addition to the complaints of residents in 2019, the existence of a reproducing widespread population was documented. In the middle of August 2019, representatives of the health department, city authorities and specialists from KABS/IfD met to discuss and to coordinate further actions. In a rst step, the residents were informed via media, and a website was created as a platform to extend reporting.
In the last week of August 2019, all households received yers and Bti-tablets for self-help. Several citizens reported severe nuisance caused by Ae. albopictus in their garden area. An action plan was designed together with authorities serving as a concept for an integrated control strategy in 2020. The plan included: 1. Press releases and information of the public by local media in close cooperation with the local authorities ( rst half of May, 2020).
2. Training of eld staff (six people) in early May including: a) the biology and taxonomy of mosquitoes to be able to distinguish between Ae. albopictus and other container-breeding mosquitoes such as Cx. pipiens s.l./Cx. torrentium, Culiseta annulata, Cs. longiareolata or Ae. japonicus; b) the breeding habitats of Ae. albopictus and how to identify and enter the breeding sites in a database; c) the correct application of the biological larvicide Bti; d) the handling of mosquito traps; and e) how to approach the residents, especially under the restrictions imposed by Covid-19.
3. Flyers with detailed information along with Bti-tablets for self-help were distributed to 1820 households on 11th and 12th of May, 2020.

Metzgergrün in Freiburg
From June to October 2020, at three-weekly intervals, accessible gardens were inspected and treated with Vectobac WG in ve rounds.

Integration of the Sterile Insect Technique (SIT)
The third pillar along with CP and DtD control was to challenge Ae. albopictus populations by sterile males under eld condition. This included the logistic of a steady supply with sterile males, the quality control and their effect on eld populations.
In this context, Ae. albopictus eggs collected in Heidelberg in the 2017, were used to start the mass-rearing at the Centro Agricoltura Ambiente "G. Nicoli" (CAA) in Crevalcore, Italy. This approach was chosen to prevent the use of mosquitoes with different genetic backgrounds.
Shipping from Italy to Germany was started following approval by the authorities.

Mass production of Aedes albopictus
An effective mass-rearing technology is essential to ensure sustainable large-scale production of high quality sterile males [31,32,33,60]. In our program, the mass-rearing methods developed by the Insect Pest Control Laboratory (FAO-IAEA) have been adopted [32][33][34][35].
Sex sorting was performed by the Fay-Morlan glass sorters at the pupal stage in water [50]. This technique exploits the difference in size between male and female pupae. The aim was to keep the number of residual females to no more than 1% of the released sterile males. for 20 minutes in each cage to allow the females to take an ad libitum blood meal. The number of biting females per offering session was recorded. The females were kept for another six days in the cages for oviposition. The wooden boards were removed and kept for ve days in chambers with wet cotton (> 90% RH) to allow for complete embryogenesis. Then, the wooden boards were transferred into a hatching container (size: 22x7x4.5 cm) and ooded with tap water. The hatched larvae were counted after 24 hours and removed from the container. Then, containers with wooden boards were lled with a 10% hydrogen-peroxide solution and kept for 48 hours at 25 ± 2°C to bleach the exochoria [52]. The boards were removed and all eggs (including the egg shells of the hatched larvae) were counted using a binocular (Motic, SMZ-171, Germany) and the embryogenesis of each single egg was assessed. Due to the resulting transparency of the exochorion in eggs with fully developed embryos, the eyes of the embryo and the "hatching tooth" could be easily recognised as dark spots on the head capsule at the anterior part of the embryo. It cannot be excluded that some of the developed embryos have chromosomal damages which doesn't allow hatching or normal development. Thus, the sterility may had been underestimated. Eggs showing no embryonic structures were rated as "sterile". The sterility was also tested by disrupting or bursting the egg-shell with a needle to identify the segmentation of an existing embryo or non-segmented whitish egg masses. Non-embryonated egg-shells burst easily when touched with the needle.

Effect of the ratio between sterile and fertile males in cage experiments
Reduction of wild Ae. albopictus populations by standard control measures prior to the application the SIT technique is essential to increase the e cacy. Even though the dose of radiation is known to damage the sperm but not the somatic cells of the sterile males [30], the sterile males could be less competitive than the wild males in the mating process. Thus, the effect of different ratios of wild males versus sterile males was tested.
The design was the same as in the previous test series. In BugDorm cages (size: 30x30x30 cm) (BioQuip, USA), 30 females per cage were challenged with the following ratios of wild to sterile males: a) 1:1 (15 wild and 15 sterile males); b) 1:5 (5 wild and 25 sterile males); c) 1:10 (3 wild and 30 sterile males. In each cage, three cylindrical dark containers (diameter: 7 cm; height: 6 cm) half-lled with water and holding a wooden board for oviposition. As mentioned above, the same carbohydrate source was offered. Alike, after 24, 48, 72, 96 120 and 144 hours, an ad libitum blood meal was offered by the forearm of the PI. The number of biting females per blood feeding session was recorded. After the last blood meal, the females were kept for another six days in the rearing cages to allow oviposition. The wooden boards were removed after six days, marked and kept for another ve days in chambers with wet cotton (> 90% humidity) to allow complete embryogenesis. The rate of embryogenesis was assessed as described in the previous experiment. Each trial was conducted in three replicates.

Shipment of sterile Aedes albopictus males
The shipment of the sterile males has to be cost-effective and timely, with the mortality rate of the caged males being as low as possible. In the course of our study, we tested the shipment in small round plastic containers

Field application of SIT a) Sector B in Melm, City of Ludwigshafen
The sterile males were released within trial area B (Melm, City of Ludwigshafen) after standard interventions by CP and DtD control. The release took place early evening at 7pm in 13 sites ( Fig. 2; Table 1). Eighteen recurrent weekly releases between the 29th of May and the 7th of October, with more than 310,000 sterile males were carried out. This corresponds to a mean number of 1,013 sterile males released per week/ha within the 17 ha trial area.  Fig. 3). The higher number of released sterile males was chosen because of the larger wild population of Ae. albopictus in the Metzgergrün district compared to the Melm district in Ludwigshafen.

Assessment of the e cacy of the implemented control strategy
The surveillance of the Ae. albopictus population was based on inspections of the breeding sites including larval sampling as well as egg counts and embryogenesis check on ovitraps.

a) Larval sampling
Breeding sites holding water were inspected for mosquitoes by aid of a torch and samples were collected with a plankton net and identi ed to the species level [38].

b) Ovitraps management
Standard ovitraps to determine the number of deposited eggs were the main tool to assess the effect of the intervention, also allowing an estimate of the population density [53]. The ovitraps consist of a dark plastic container with a total volume of 1.5 litres. They were positioned on the ground or hung on shaded places at a maximum height of 1.5 metres and lled with hay infusion (3 g hay pellets dissolved in 5 l of tap water) up to a level of 3/4. A wooden board (length: 17 cm; width: 3 cm) was added to support oviposition. In order to prevent the development of larvae to adults, 10 grains of Vectobac G (activity: 200 ITU/mg, Valent BioSciences, Libertyville, USA) were added to the water. The wooden boards were replaced at bi-weekly intervals and the water was replenished. The boards were marked with the date and site of collection, wrapped in paper foil and stored in a refrigerator until egg count. Random morphological determination was done by hatching some of the eggs and rearing to the fourth larval instar [37,38]. Sterile eggs were identi ed as described above.
Deployment of the ovitraps in the test areas a. Melm: Starting on 18th of May 30 ovitraps were evenly positioned across the 65 ha area, ten in each of the three sectors A, B and C (Fig. 2). The wooden boards with the eggs were collected bi-weekly between the 1st of June and 19th of October 2020. The number of eggs and embryogenesis were assessed as described above (Fig. 2).
b. Metzgergrün (Freiburg): Across the area of 4.5 ha, 21 ovitraps were positioned as described above. This district was heavily infested with Ae. albopictus, and therefore chosen as the SIT test area. Following DtD control and application of Bti (Vectobac WG), the release of sterile males started in middle of July (Fig. 3).
c. Gartenstadt (Freiburg): this district was chosen as control area without SIT application. It was similarly infested by Ae. albopictus as Metzgergrün. Eighteen ovitraps were installed. The wooden boards were collected bi-weekly between 15th of August and the 13th of October. The number of eggs and the sterility were assessed as described above.

Statistical analyses
For statistical analyses, the Student's t-test (two sample assuming unequal variances) was applied to the quality control of sterile males test to evaluate the sterility of the eggs and to the cage experiments to assess the effect of the ratio sterile to fertile males (Microsoft Excel, version 16. 45,21011103) 3. Results

Community participation
A total of 1,820 households received written information on the control of Ae. albopictus and Bti-tablets in their mailboxes (less than one minute/household). The positive effect of this activity and the active cooperation of the residents is documented by the high rate permitting access to the houses during the rst DtD round at the end of May/early June amounting to 78.38% access; only 9.13% refused entry to their properties, while 12.49% were absent. More than half of the properties (55.42%) did not contain any breeding sites (   Fig. 4. Mortality of 100% was achieved during eight weeks, followed by a small decrease (of 1%) over the next four weeks. Only after 15 weeks mortality started to drop below 90%. These results were obtained with a weekly exchange of 25% of the water to simulate the use of collected rainwater to irrigate gardens.
Larval control of Ae. albopictus in large containers was achieved for a whole month with an initial application rate of (6.75x10 6 ITUs/container) as described in 2.2.2.1 (Fig. 5).

E cacy of the DtD treatments in Melm, Ludwighafen
During the ve DtD rounds, including two complete rounds (round 1 and 5) and three hotspot rounds (rounds 2-4) at properties and the surroundings when positive ovitraps or mass breeding sites have been observed, a total of 2.683 properties have been inspected. Detailed results are given in Table 2. Out of the 2.683 properties, 2.035 (75.85%) could be entered, while 418 (15.6%) owners were absent and 230 (8.57%) refused permission to enter. A total of 6.328 breeding sites including 83 rainwater containers were treated with a high Bti dosage to achieve a long-lasting effect and recorded in a Q-Gis programmes. Out of the 6.328 breeding sites 129 were infested with mosquito larvae (CI: 2%) and 18 were infested with Ae. albopictus (CI: 0.28%). The highest CI values were recorded end of August with 4.41% for all mosquitoes and 0.88% for Ae. albopictus ( Table 2). The positive effect of the control strategy is documented with a CI of 0.49% for Ae. albopictus end of September. A total of 1.169 working hours of the two to six inspectors have been invested which results in an average inspection time/property of 26 minutes. Altogether 17.3 kg of Vectobac WG were applied to the 2.683 breeding sites.

E cacy of routine DtD treatments in Metzgergrün, City of Freiburg
The routine treatments in Metzgergrün, City of Freiburg, were performed over ve rounds of DtD activities and Bti-treatments. Starting in the second half of June, the routine treatments were conducted in a three-week rhythm. The Bti-application was carried out until all properties were treated, lasting ve consecutive days. After each application round, the accessibility of the area was evaluated and recorded. Compared to other areas in the city of Freiburg, the accessibility of the properties was classi ed as ranging from poor to moderate.
Throughout the season, residents were provided with blisters of Bti-tablets for self-help including those whose properties could not be inspected. The mean egg sterility of females, inseminated by sterile Ae. albopictus males amounted to 87.53 ± 9.15%, whereas the sterility of eggs derived from non-irradiated males and females was only 3.3 ± 2.8% (Table 3). The real sterility might be even higher because the bleaching method may fail in detecting embryos going through delayed mortality (data not published). According to the Student's t-test, all results are signi cant (p ≤ 0.001). In contrary, 96.70 ± 2.8% of the eggs derived from non-irradiated individuals were embryonated, while only 12.47 ± 6.49% of the eggs laid by females inseminated by radiated males developed into embryos (Table 3).

Accuracy in sex sorting
The number of radiated Aedes-females in the delivered batches slightly exceeded 1%. The number of 1.13 ± 0.64% recorded by CAA is almost identical to the numbers (1.19 ± 0.63%) evaluated by IfD (Table 1). These data indicate a release of about 3,700 radiated females in Ludwigshafen (Melm) and 1,620 radiated females in Freiburg (Metzgergrün).

Determination of the most effective ratio between sterile and fertile males in cage experiments
The results of this experiment document the importance of the determination of the ratio between wild and radiated males (Fig. 6). The egg sterility of the non-irradiated populations amounts to 5.9 ± 6.6%. When the ratio between radiated and non-irradiated males is 1:1 ( In Table 1 (Table 4). See also Supplement to Table 4  The weekly release of about 1,000 sterile males/ha from early May until early October in the SIT area (section B) resulted in an overall sterility of 82.62% (Table 4). The sterility of the 274 eggs collected in section A was 43.8%, and of the 220 eggs collected in section C was 38.64%. The relatively high sterility in the adjacent non-SIT sections A and C can be explained by the migration of sterile males and/or females mated with sterile males into the non-SIT control areas. The fact that no eggs were recorded in any of the ovitraps after the 7th of September indicates the successful control strategy.
In the second half of September, 2,668 breeding sites were inspected and only 13 breeding sites contained the larvae of Ae. albopictus. resulting in a very low CI albo of 0.49%.

Freiburg
The 21 ovitraps employed during the six bi-weekly sampling period in the SIT area in Freiburg (Metzgergrün) from the 14th of July ( rst collection date: 28.07.2020) until the 13th of October collected a total of 2,298 eggs (Table 5), which was about six-times higher when compared to almost the same time period in the SIT area in Ludwigshafen. The release of about 2,320 sterile males per hectare on a weekly basis from the 15th of July to the 7th of October produced sterility rates increasing from 14.9% on the 28th of July to 96.6% on the 13th of October (Fig. 7) The mean sterility was 77.4 ± 15.35%.
In the control area (without SIT) (Freiburg Gartenstadt) on the collection dates from 29th of August to the 13th of October (4 x bi-weekly sampling), 1,615 eggs were collected (

Discussion
The goal of this large-scale integrated eld trial was the maximum reduction or even the elimination of established populations of Ae. albopictus. In addition to the two pillars CP and DtD control by trained staff with long lasting Bti-treatment, the SIT was used as the third pillar. CP and DtD are important to reduce Ae. albopictus in obvious and easily accessible breeding places. Sterile males are able to nd remaining females in hidden mating places. The selected trial sites, infested by Ae. albopictus in the urban environments of the cities of Ludwigshafen and Freiburg, were ideal for the three-pillar approach. The majority of Ae. albopictus breeding sites are located on private properties, thus it must be in the own interest of the residents to prevent breeding of Ae. albopictus in close proximity. If properly instructed and using Bti, the collaboration with residents is costeffective and sustainable [25,26,43]. The support by DtD activities conducted by trained staff proved to be the backbone of the integrated control approach. The cooperation between the residents and the DtD teams still leaves room for improvement since the minimum of 95% of accessible properties was not achieved [29]. The mean costs involved in the control measures by CP and DtD control amounted to € 2.50 per property.
The control of the container-breeding Ae. albopictus requires a long-lasting larvicides to make it cost effective and e cient. This goal was achieved with increased doses of Bti [45]. The results obtained in Ludwigshafen were convincing. As a result of the stringent Bti application the container index of Ae. albopictus was pushed down from 10.9% to below 1%. (Table 2). In contrast to the previous year, no nuisance by residents was reported. This is also underlined by the low number of eggs found in the ovitraps in the Melm area (mean number/ovitrap 4.3). In the control area Freiburg Gartenstadt six ovitraps contained considerably more than 100 eggs/ovitrap and a maximum of 340 eggs/ovitrap/two weeks. Carrieri et al. [49] calculated the epidemic risk threshold for a Chikungunya transmission based on the mean egg density/trap/week. Infections can already occur when the average number of eggs/trap/week is above 44 eggs. In the control area of Gartenstadt this threshold was exceeded. The climate change with rising temperature will very likely accentuate this problem.
Risk-bene t parameters do not apply to the integrated approach for the control of Ae. albopictus. The use of Bti even in higher doses is safe and the release of sterile males do not bear any negative impact. The cost-bene t factor needs of course special consideration. In general, the costs are an up-front investment to prevent spread and high density of Ae. albopictus populations. Protection from autochthonous viral infections must have the highest priority. Thus, the costs to protect from Ae, albopictus amount to an estimated € 2 per person per season. This is highly cost effective considering the severe potential implication for public health.
Germany, like other countries north of the Alpes, are at the beginning of the invasion by Ae. albopictus, and thus confronted with a big challenge. Containment of Ae. albopictus will only succeed by close collaboration between all the stakeholders, from the directly affected residents to the eld workers, researchers, up to government agencies. [39]. The importance and urgency to combat the Asian tiger mosquito has been recognized by all levels of the Federal Republic of Germany. Programs for surveillance, risk assessment and the evaluation of control tools are actively supported by the Federal Agency for Environmental Protection (UBA) in cooperation with the Friedrich Lö er Institute (FLI) and the Bernhard Nocht Institute (BNI). The State of Baden-Württemberg, which is currently most affected by Ae. albopictus, nanced risk assessment programs concerning the establishment of Ae. albopictus on a community level [24].
The toolkit to combat container-breeding mosquitoes includes powerful techniques and strategies like community participation, microbial control tools (e.g. Bti), insect growth regulators, surface layers, the sterile insect technique (SIT) e.g. based on radiation, the insect incompatibility technique through the endosymbiont Wolbachia-induced incompatibility (IIT), or the combination of both SIT and IIT [29,38,40]. Last but not least, chemical products such as methoprene, di ubenzuron, pyriproxyfen (IGRs) or pyrethroids as adulticides can be applied in case of outbreaks only. Space spraying of adulticides should be only applied in the case of health emergencies [41]. Modern genetic approaches as in site-speci c gene editing with CRISPR/Cas9 can augment the currently existing toolbox. Cas9-mediated gene-editing can be an e cient platform for gene-driven strategies to introduce suppression and pathogen-blocking genes into wild mosquito populations [38,42].

Conclusions
This eld pilot trial shows that an integrated control program is most effective when it comprises three pillars, namely a) community participation, b) DtD activities including long-lasting Bti-larviciding of all accessible larval sites to strongly reduce wild Aedes albopictus population as a precondition a successful application of SIT, and c) SIT to strongly reduce or even eradicate Ae. albopictus populations. The combination of Bti and SIT, two highly effective, selective and safe measures may allow the achievement of Ae. albopictus suppression without any negative impact on the public health and the environment.
Declarations Figure 1 Recorded Aedes albopictus populations in 2019 (red: not under control; green: eradicated; green-red: strongly reduced; yellow: population size not known).
Page 27/31  Long-term effect of Vectobac WG on Aedes albopictus applied to large water containers. Effect of the ratio between radiated and non-irradiated males on the egg sterility rate. (see also Supplement 4 to