2.1 Study areas
The study was conducted in two Ae. albopictus infested areas in Southern Germany: a) The “Melm” district in the city of Ludwigshafen (Palatine) and b) the “Metzgergrün” district in the City of Freiburg (Baden-Württemberg). The “Gartenstadt” district in Freiburg served as a control area to the SIT area “Metzgergrün”.
a) Melm is a new district of Ludwigshafen which was built at the end of 1990 and in the early 21st The total size is about 65 hectares and it is embedded in a green area with lakes for recreation. It consists of about 950 estates with single family and double houses, as well as some apartment buildings. Almost every house possesses an adjacent garden area which can provide potential breeding sites for Ae. albopictus. The area is well defined and well suited as a test area due to the complaints of the residents about nuisances caused by Asian tiger mosquitoes.
The study area of about 65 ha was divided in three sectors of almost equal size, each with specific mosquito control actions: Sector A: 23 ha (CP+DtD); Sector B (CP+DtD+SIT): 17 ha; and Sector C: 25 ha (CP+DtD) (Fig. 2).
b) Metzgergrün in Freiburg. The district has a size of 4.5 ha and is mainly characterised by apartment buildings with social housing along 4 streets (Fig. 3). This district was chosen to employ the SIT technique in addition to CP and DtD Bti treatments because this residential area includes a large portion of gardens adjacent to apartments/residential houses which include a vast number of breeding sites like used tires and garbage. The garden plots were usually locked and the owners were unknown. Frequently, the inhabitants were not cooperative, and it was difficult to enter the gardens to treat the breeding sites on a three-weekly interval from July until October 2020 as was done in the rest of the albopictus infested area in Freiburg.
c) Gartenstadt (Freiburg): This district of Freiburg is about 1 kilometre away from the SIT-area (Metzgergrün) and similarly infested by albopictus as Metzgergrün, therefore, this area was chosen as a control area where CP and DtD were deployed as well as ovitraps to assess the sterility of an SIT untreated area in comparison to Metzgergrün.
2.2 Assessment of the efficacy of the “three column strategy” including Community participation, DtD activities with Bti treatments and SIT
2.2.1 Community Participation
In the first 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 fight against Ae. albopictus. Between the 9th and 12th of May, three people distributed flyers to 1820 households with detailed information for the help of residents to control the Asian tiger mosquito on their properties. Each household received a flyer to which a paper box containing 10 Bti-tablets in a blister pack (Culinex® Tab plus, Lot: 0604783, activity: 1000 ITU/mg, Culinex GmbH, Ludwigshafen) including a package insert with instructions for application was attached by organic glue. The Bti-tablets were sterilised and contained no spores or living bacteria [44]. They had a weight of 550 mg/tablet and one tablet was enough to treat 50 l of water [14]. It was recommended that the treatment be repeated on a fortnightly interval.
Additionally, people were informed about the characteristics of Ae. albopictus and asked to record suspicious biting mosquitoes by killing them or making them immobile with a slight touch during biting. People were asked to send the mosquitoes to our laboratory or take a picture with a mobile phone showing the hind legs, head, or thorax for species determination. 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 scope of CP. Furthermore, the residents of the estates were interviewed about their knowledge of control activities. During the last control round (no. 5) in September, the residents were asked if they had implemented the proposed control activities on their property by applying the Bti-tablets or environmental sanitation.
2.2.2. Assessment of the effect of Bti-treatments
Whereas the control of floodwater mosquitoes requires only a minimum effective dosage of Vectobac WG (strain AM5265, Valent Biosciences, Libertyville, USA), to achieve a knock-down effect of the floodwater mosquito larvae, which hatch during a flood [56], the control of container-breeding mosquitoes needs a long-term effect due to the usually constant follow-up of generations. Thus, the sequence of retreatments and the costs for manpower could be strongly reduced when a long-term effect is achieved.
In our routine control program against the third and early fourth instars of floodwater mosquitoes, we applied 500 g Vectobac WG (potency when sterilized 2,700 ITU/mg) which resulted in a dosage of 0.5 mg/l (1,350 ITUs/l) calculated to a depth of 10 cm of the breeding site. For the control of container-breeding mosquitoes, alongside the 0.5 mg/l concentration, we tested 10x (5 mg/l) and 100x (50mg/l) higher dosages to achieve the desired long-term effect.
2.2.2.1. Efficacy of Bti-treatment in large water containers (rainwater containers)
In a first series of tests, the effect of Bti-treatments in rainwater containers was simulated. The Bti mixture was prepared with sterilised Vectobac WG (Valent BioSciences, Libertyville, USA, lot: 294458PG00, manufactured Oct. 2018). Here, 250 grams of Vectobac WG was thoroughly mixed with 1.5 l of tap water (pH: 7.8; conductivity: 680 µS). Aliquots of the suspension were taken with an Eppendorf pipette by constant stirring of the suspension and applied to plastic buckets filled with 20 litres of tap water (1/10 of the usual water volume of a regular rainwater container) to achieve the desired dosage of 1 gr (6 ml of Vectobac WG solution), 0.1 gr (0.6 ml Vectobac WG solution), and 0.01gr (0.06 ml of Vectobac WG solution) per 20 l of water. Each dosage was tested in four replicates at a constant temperature of 24°C±1°C, with 4 buckets serving as a control. At the beginning of the test series, 20 third instar larvae from a laboratory-reared Ae. albopictus colony were introduced in each bucket. The mortality reading was taken at 24 and 48h after the release of the larvae. On a weekly basis, 5 l of water was taken from each container and replaced again with fresh tap water to achieve a total water volume of 20 l and simulate natural conditions when water is removed for watering the garden. Then, 20 third instar larvae were again released in each container and the mortality reading was performed after 24 and 48 h. At each 48h reading, the larval cadavers and living larvae were counted and removed. The test was conducted until the mortality rate was less than 60%.
2.2.2.2. Efficacy of Bti-treatment in small water containers
In a second series of tests, the efficacy of Vectobac WG in four different small water containers typically found in garden areas was assessed: a) terracotta flowerpots with rough surfaces (volume: 1400 ml); b) terracotta pots – with smooth walls (volume: 1400 ml); c) plastic flowerpots (volume: 950 ml); d) zinc pots (volume: 800ml); and e) terracotta flowerpot saucers (volume: 200 ml). Before application, the containers were scrubbed with flower soil (Compo Sana potting soil) and cleaned with water and dried for 24 h to simulate natural conditions before the application of Vectobac WG (lot: 294458PG00, manufactured Oct. 2018). Four of each type of container were homogenously sprayed by means of a Mesto pressurised sprayer (Mesto BUGSI 1.5L), with 15 ml of the Vectobac WG stock solution (250 g Vectobac WG mixed with 1.5 L of tap water) resulting in 2.5 g Vectobac WG/small container. The containers were dried for 48 hours, filled with tap water, and then 20 Ae. albopictus third instar larvae were added to each container. The mortality reading was performed 48 h after the release of the larvae. The containers were emptied after the mortality reading and dried again for 48 hours to fill the containers again with water and release new batches of larvae. The procedure was repeated until the mortality rate was less than 60%. Four containers of each type served as an untreated control. The test was conducted at 24°C±1.5°C and 80% RH.
2.2.2.3. Routine Bti-Treatments of the infested areas
Melm in Ludwigshafen: The first records of biting Ae. albopictus females were obtained in early August 2019. After species identification was confirmed, the health department of the county and the city authorities were informed. Immediately afterwards, the occurrence of a reproducing Aedes population was proven on the following days via the inspection of breeding sites along 4 streets equally distributed in the Melm district. Scattered across the district, several breeding sites (unused flowerpots, saucers, rainwater barrels) were proven to be positive for the developmental stages of Ae. albopictus. In total, 55 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 further actions. In the first step, the residents were informed via media, and a website was created as a platform to record more specimens reported by the residents. In the last week of August 2019, all households received flyers and Bti tablets for self-help. After the distribution of flyers, several citizens sent emails and reported a severe nuisance caused by Ae. albopictus in their garden area. Therefore, an action plan was designed in cooperation with authorities for 2020, which should serve as a proof of concept for a successful integrated control strategy. The plan includes:
- Press releases and information of the public by local media in close cooperation with the local authorities (first half of May, 2020).
- Training of field staff (6 people) in early May. The training course includes detailed information on: a) the biology and taxonomy of mosquitoes to be able to answer the questions of residents and to distinguish between albopictus and other container-breeding mosquitoes such as Culex pipiens s.l./Cx. torrentium, Culiseta annulata, Cs. longiareolata or Ae. japonicus; b) the breeding habitats of Ae. albopictus and how to identify and record the breeding sites in a database; c) the characteristics and appropriate management of the biological larvicide Bti; d) the handling of mosquito traps; and e) how to approach the residents, especially under the consideration of Covid-19.
- The creation and distribution of flyers with detailed information and Bti-tablets for self-help (11th and 12th of May, 2020, 1820 households, 2 people, each 8 working hours).
- Deploying 30 ovitraps (⁓1 trap/2ha) more or less equally distributed in the district and inspected on bi-weekly intervals. Eggs were counted and checked for embryogenesis according to the description above (Fig. 2).
- DtD activities from May to September 2020 in five different rounds to make the action effective and cost-efficient.
- 1st round from 18th of May to 7th of June carried out by one team of two people: visiting all properties to carefully map and treat all potential breeding sites. Recording the occurrence of breeding sites, mosquito larvae and species in a Q-GIS-database is the basis for the decision-making process of the following control steps focusing on hotspots of breeding sites and the number of eggs in the ovitraps. The control of hotspot areas based on an efficient surveillance system contributes to a cost-effective reduction of the Aedes
- 2nd round from 23rd of June to 7th of July carried out by one team of two people: hotspot treatment for all properties where breeding sites of albopictus were recorded in the first round.
- 3rd round from the 28th to 31st of July carried out by one team of two people: hotspot treatments of breeding sites in properties over a 100 m radius of the 6 ovitraps (2A, 4A, 5A, 1B, 8B, 9B) in which eggs of Aedes albopictus were found in ovitrap collections at 29th of May, 15th of June, 29th of June and 13th of July.
- 4th round from the 17th to 21st of August (one team): similar to the 2rd round, inspections and treatments of properties with rainwater containers and small breeding sites.
- 5th round: Similar to the first round. All houses were inspected, and breeding sites were controlled for larvae and treated. Larvae were collected and determined by species in the laboratory. Additionally, a questionnaire was prepared and those residents who agreed to participate were asked about their own contribution to combat tiger mosquitoes and their view of the success of the ongoing control activities (three teams each of two people (14th to 22nd of September).
Metzgergrün in Freiburg: From June to October 2020, more or less in a three-weekly interval, accessible gardens were inspected and treated with Vectobac WG in 4 rounds as was performed over the rest of the infested area in the City of Freiburg.
2.2.3. Application of the Sterile Insect Technique (SIT) against Aedes albopictus
One of the major goals of the study was the assessment of the effectiveness and efficiency of the SIT approach against Ae. albopictus under routine conditions, including two different transport systems in a routine delivery practice, the quality control of the released male populations and the effect on the sterility of the eggs laid by the wild population.
In this cooperative program, Ae. albopictus derived from our German Aedes albopictus population in Heidelberg was mass-reared at the Centro Agricoltura Ambiente “G. Nicoli” (CAA) in Crevalcore, Italy, and irradiated in the nearby hospital in Ferrara. Several hundred eggs were collected in ovitraps at the Heidelberg population in 2017 and sent to CAA to establish a stock colony. This approach was adopted to prevent the introduction of mosquitoes with different genetic backgrounds from the local one.
2.2.3.1. Mass production of Aedes albopictus at CAA
An effective mass-rearing procedure is essential to ensure the large-scale production of quality sterile males over a long period [31,32,33]. The rearing process should not lead to the artificial selection of genetically homogeneous individuals whose competitiveness with wild individuals is significantly reduced due to the loss of fitness. Therefore, with the support of the Insect Pest Control Laboratory (FAO-IAEA) in Seibersdorf, Austria, mass-rearing technologies have been developed and employed in our program.
Following authorisation obtained from the local governments (Regierungspräsidien) in Neustadt (State of Palatine) and Freiburg (State of Baden-Württemberg), the mass production of Ae. albopictus was started at the CAA facility from eggs collected in Heidelberg. Mass production was conducted by applying procedures as previously published without using any animal as a blood supply [32-35].
2.2.3.2. Sex sorting
Sex sorting was performed by the Fay-Morlan glass sorters on the pupal stage in water [50]. This technique takes into account that the male pupae are significantly smaller that the female pupae. CAA aims for no more than about 1% of females per released batch of sterile males.
2.2.3.3. Sterilisation by pupal irradiation
Sterilisation was conducted at the Medical Physics Department of St. Anna Hospital (Ferrara, Italy) by an IBL 437 irradiator (CIS Bio International, Bagnols-sur-Cèze, France) with a Cs-137 linear gamma ray source, exposing male pupae aged 24-32 h in water [30]. After dose-response studies, the pupae were routinely radiated at a dose of 35 Gray (1.85 Gy/min for 19 minutes). In previous tests it was shown that this dosage provides the best results concerning the male sterility and competitiveness of sterile males versus fertile, wild males [30,36].
After irradiation, the pupae were kept in a climatic room at 28 ±1°C for adult male emergence, after which adults were chilled at 8-10°C and packaged for delivery to the field site by DHL flight express service. Time from exiting the production facility to the release areas was always below 24 hrs.
2.2.3.4. Quality control of the sterile Aedes albopictus males
Random samples of thirty sterile males were withdrawn from three different batches (SIT-Batches: 7, 8, 9; see Tab. 1) and released each in a Bug Dorm rearing cage (BioQuip, CA, USA). Virgin Ae. albopictus females were derived from our colony (Heidelberg strain) by separating individual pupae in small glass vessels covered with a net to allow single specimens to emerge, thus guaranteeing that females are not exposed to males and inseminated before the test. The emerged females in each glass vessel were checked macroscopically by the less plumose antenna, shorter palps and proboscis. In each of the three cages, 30 unirradiated virgin females were introduced to the 30 radiated males and kept at 25±2°C and 70±5% RH (dark: 8 h/light: 16 h). Three cylindrical dark containers (diameter: 7cm; height: 6 cm) were positioned in each cage, half filled with water and holding a wooden board (size: length: 8 cm; width: 3cm) as a substrate for egg-laying. In addition, a vessel with cotton soaked with a 10% sugar solution, 10 raisins and a piece of apple was offered as a carbohydrate source. Three similar cages with the same number of unirradiated males and females derived from the laboratory colony were kept in parallel under the same conditions as the control. After 24, 48, 72, 96, 120 and 144 hours, the lower arm of the PI was offered for 20 minutes in each cage to allow the females to take a blood meal until ad libitum. 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 marked and removed and kept for five days in chambers with wet cotton (>90% humidity) to allow for complete embryogenesis. Then, the wooden boards were transferred into a hatching container (size: 22x7x4.5cm) and flooded with tap water. The hatched larvae were counted after 24 hours and removed from the container. Then, containers with wooden boards were filled with a 10% hydrogen-peroxide solution and kept for 48 hours at a temperature of 25±2°C to bleach the exochorion of the eggs [52]. The single boards were removed and all eggs (including the egg shells of the hatched larvae) were counted using a binocular (Motic, SMZ-171, Germany); 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. Eggs showing no embryonic structures were addressed 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.
2.2.3.5. Effect of the ratio between sterile and fertile males in cage experiments
A sufficient reduction of an established wild Ae. albopictus population is essential for the successful employment of the SIT technique. In the mating process, the sterile males have to compete with the wild males, which are supposed to be more vitally performing than the radiated males; even the dose of radiation is known to damage the sperm but not the somatic cells [30]. In this experiment, the effect of different ratios of wild males versus sterile males was tested. The test design was the same as in the previous test series. In bug dorm cages, the following ratios of wild to sterile males together with 30 unirradiated females were evaluated: a) 1:1 (15 wild and 15 sterile males/cage); b) 1:5 (5 wild and 25 sterile males/cage); c) 1:10 (3 wild and 30 sterile males/per cage). In each cage, 30 unirradiated females were released after the release of males. In each cage, three cylindrical dark containers (diameter: 7cm; height: 6cm) half-filled with water and holding a wooden board as substrate for egg-laying, were positioned. In addition, a vessel with cotton soaked with a 10% sugar solution, 10 raisins and a piece of apple was offered as a carbohydrate source. Following the release of the females after 24, 48, 72, 96 120 and 144 hours, the lower arm of the PI was offered for 20 minutes in each cage to allow the females to take a blood meal ad libitum. 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 full oviposition. The wooden boards were removed after six days, marked and kept for another five days in chambers with wet cotton (>90% humidity) to allow complete embryogenesis. The rate of embryogenesis was assessed as described in the previous experiment by bleaching and bursting the egg shell to control the segmentation or non-segmentation of the embryo or egg mass.
2.2.3.6. Shipment of sterile Aedes albopictus males
The shipment of the sterile males has to be cost-effective and timely, and the stress (mortality rate) of the caged males has to be as low as possible. In the course of our study, we tested two types of small containers for the shipment of radiated males: a) shipment in small round plastic containers (diameter: 5.2cm; height: 4.7cm) covered with a dark plastic lid; or b) shipment in plastic tubes (height: 45 cm; diameter: 8cm) covered with nets and fixed with a rubber band. Each small container holds about 1000 radiated males. The small round plastic containers were packed in an outer styropor box (size: 49x36x36cm, volume 63.5 L) which contained 11 gel-cooling elements (Blu Ice, DRYCE; www.dryce-pharma.com) and two frozen (Green Ice, DRYCE) to keep the temperature in the styropor box at approximately 10°C. The frozen cooling elements were in bubble foil to avoid straight contact with the containers holding the sterile males. The outer styropor box (size: 48x99x39cm: 185.3 L) holding the plastic tubes had to be bigger because of the larger size of the plastic tubes compared to the small round plastic containers. According to the size of the treated area, the styropor box contained up to 30 small plastic containers or plastic tubes, respectively. The styropor boxes were shipped on a weekly basis from May until October by DHL on an overnight service. The cost of each shipment with DHL was recorded.
2.2.3.7. Assessment of the accurate number of the sterile males and females per container and shipment
Out of the 18 shipments from CAA, Italy, one plastic container holding approximately 1,000 radiated males was transferred to a refrigerator and kept for 2 hours at -15°C to kill all mosquitoes. With the aid of a binocular (Motic, SMZ-171, Germany), the number of males and females per container was determined. Thus, the average number of released males could be determined. Furthermore, it was our goal to keep the contamination of the samples with Aedes females as low as possible with the appropriate use of the sexing technique [50]. The percentage of females per small container should not exceed 1% in order to not contribute to a nuisance situation caused by released radiated Aedes females.
2.2.3.8. Field application of SIT
a) Sector B (CP+DtD+SIT) in Melm, City of Ludwigshafen
During the course of the study, in sector B, in addition to the DtD activities and the application of Bti, the SIT approach was applied. The results were validated in comparison to districts A and C, which served as a control. The styropor boxes containing the sterile males were shipped weekly overnight on Mondays by DHL and arrived in our laboratory on Tuesday at midday. The release took place in the late afternoon at around 7pm at 13 release spots (Fig. 2). Before the release, the containers holding the sterile males were kept at room temperature to acclimatise the males.
Altogether during 18 releases between the 29th of May and the 7th of October, more than 310,000 sterile males amounting to 320 containers were released. The number of containers per release varied between 12 and 30 depending on the availability of radiated males at CAA (Table 1). Considering the size of sector B (SIT area) of 17 ha, a mean number of 1,013 sterile males/ha was released from 29th of May until 7th of October on a weekly basis.
a) Metzgergrün, City of Freiburg
From the 15th of June to 7th of October 2020, 136,000 sterile males were released homogenously over the 4.5 hectares, which amounts to 2,320 sterile males/hectare altogether (Tab. 1; 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.
2.2.3.9. Assessment of the efficacy 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 the employment of ovitraps.
a) Larval sampling
Breeding sites holding water were inspected for mosquito development stages, either by employing a plankton net in larger water containers or pouring the water through a plankton net when small breeding sites were examined. In larger water containers like rainwater barrels, the net was drawn through the water in a figure of “8” pattern to sample the larvae. By the aid of touch, the mosquito developing stages could be easily identified in the water column.
For transportation to the laboratory, a plastic container with a close-fitting cap was 3/4 filled with water from the site. The containers were carefully marked by recording the date and location of sampling for the determination of container indices. The third and fourth instar larvae were identified to the species level by using the keys in Becker et al. [38]. Earlier larval instars were reared to the 4th instar by a mosquito breeder for species determination.
b) Employing Ovitraps
Ovitraps were the main tool with which to monitor the presence, phenology and abundance of Ae. albopictus and to assess the effect of control activities as well as to estimate the population density based on the number of deposited eggs [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. They were filled 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. The board or wooden stick had a rough and smooth side and was positioned with the rough side facing upwards to provide small depressions on the surface of the board which are ideal for eggs to be deposited in. In order to prevent the potential 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 a bi-weekly interval and the water in the ovitraps was refilled. The collected wooden boards were clearly marked with a permanent marker at the dry end of the stick to refer to the location of the ovitrap and the date of collection. They were kept in paper foil and stored in a refrigerator until they were checked by means of a binocular (Motic, SMZ-171, Germany) for the presence of eggs.
A skilled person is able to distinguish between eggs of the indigenous species Ae. geniculatus and the exotic species Ae. albopictus, Ae. koreicus and Ae. japonicus. The results have been validated by hatching some of the eggs and rearing the larvae to the fourth instar for morphological determination [37,38].
The sterility of eggs was checked as described above by bleaching the exochorion of the eggs using a 10% hydrogen peroxide for 48 hours and disrupting the egg-shell to prove existing embryos or unsegmented whitish egg masses.
Employment of the ovitraps in the test areas
a) Melm: on 18th of May across the whole area of 65 ha, 30 ovitraps were positioned at almost equal distances at half-shadowed spots on the ground, namely in sector A: 10 traps; B (SIT): 10 traps; and C: 10 traps (Fig. 2). Each of the ovitraps covered about 2 ha of the test area. The wooden boards were collected in bi-weekly intervals from the 1st of June until the 19th of October 2020. The number of eggs and embryogenesis were assessed as described above (Fig. 2).
b) Metzgergrün (Freiburg): on 14th of July, across the area of 4.5 ha, 21 ovitraps were installed as described above. The district was heavily infested with albopictus and, therefore, chosen as the SIT test area. Beside the DtD application of Bti (Vectobac WD) in this district, the sterile insect technique was additional applied from the middle of July onwards (Fig. 3).
c) Gartenstadt (Freiburg): this district is similarly infested by albopictus area as Metzgergrün; therefore, this area was chosen as a control area without the employment of SIT. Eighteen ovitraps were set up the 15th of August and the wooden boards were collected in a fortnight interval until the 13th of October. The number of eggs and the sterility were assessed as described above.
2.3. Statistical analyses
For statistical analyses, the Student’s t-test was applied by Microsoft Excel, version 16.45 (21011103).