A fundamental step in establishing a mass production system is the development of a larval diet that promotes high adult performance at a reasonable

Background: The invasive species Aedes albopictus, commonly known as the Asian tiger mosquito, has undergone extreme range expansion by means of steady introductions as blind passengers in vehicles traveling from the Mediterranean to south-west Germany. The more than 25 established populations in the State of Baden-Württemberg, Palatine and Hesse (south-west Germany) have become a major nuisance and public health threat. Aedes albopictus deserves special attention as a vector of arboviruses, including dengue, chikungunya and Zika viruses. In Germany, Ae. albopictus control programs are implemented by local communities under the auspices of health departments and regulatory offices. Methods: The control strategy comprised three pillars: (i) community participation (CP) based on the elimination of breeding sites or improved environmental sanitation, using fizzy tablets based on Bacillus thuringiensis israelensis (fizzy Bti tablets; Culinex® Tab plus); (ii) door-to-door (DtD) control by trained staff through the application of high doses of a water-dispersible Bti granular formulation (Vectobac® WG) aimed at achieving a long-lasting killing effect; and (iii) implementation of the sterile insect technique (SIT) to eliminate remaining Ae. albopictus populations. Prior to initiating large-scale city-wide treatments on a routine basis, the efficacy of the three elements was evaluated in laboratory and semi-field trials. Special emphasis was given to the mass release of sterile Ae. albopictus males. Results: More than 60% of the local residents actively participated in the first pillar (CP) of the large-scale control program. The most effective element of the program was found to be the DtD intervention, including the application of Vectobac® WG (3000 ITU/mg) to potential breeding sites (10 g per rainwater container, maximum of 200 l = maximum of approx. 150,000 ITU/l, and 2.5 g per container < 50 l) with a persistence of at least 3 weeks. In Ludwigshafen, larval source management resulted in a Container Index for Ae. albopictus of < 1% in 2020 compared to 10.9% in 2019. The mean number of Aedes eggs per ovitrap per 2 weeks was 4.4 in Ludwigshafen, 18.2 in Metzgergrün (Freiburg) (SIT area) and 22.4 in the control area in Gartenstadt (Freiburg). The strong reduction of the Ae. albopictus population by Bti application was followed by weekly releases of 1013 (Ludwigshafen) and 2320 (Freiburg) sterile Ae. albopictus males per hectare from May until October, resulting in a high percentage of sterile eggs. In the trial areas of Ludwigshafen and Frieburg, egg sterility reached 84.7 ± 12.5% and 62.7 ± 25.8%, respectively; in comparison, the natural sterility in the control area was 14.6 ± 7.3%. The field results were in line with data obtained in cage tests under laboratory conditions where sterility rates were 87.5 ± 9.2% after wild females mated with sterile males; in comparison, the sterility of eggs laid by females mated with unirradiated males was only 3.3 ± 2.8%. The overall egg sterility of about 84% in Ludwigshafen indicates that our goal to almost eradicate the Ae. albopictus population could be achieved. The time for inspection and treatment of a single property ranged from 19 to 26 min depending on the experience of the team and costs 6–8 euros per property. Conclusions: It is shown that an integrated control program based on a strict monitoring scheme can be most effective when it comprises three components, namely CP, DtD intervention that includes long-lasting Bti-larviciding to strongly reduce Ae. albopictus populations and SIT to reduce the remaining Ae. albopictus population to a minimum or even to eradicate it. The combined use of Bti and SIT is the most effective and selective tool against Ae. albopictus, one of the most dangerous mosquito vector species. Graphical

Aedes albopictus, an invasive mosquito species, is currently well established in many European countries, posing high risks to human health. A preliminary trial using repetitive releases of irradiated sterile male mosquitoes was designed, implemented and evaluated for the first time in Greece. The main scope of this trial was to investigate the efficacy of sterile insect technique (SIT) on wild population egg hatch rate in Greece using mass-produced sterile male mosquitoes from another country (Italy). The study was conducted in Vravrona area, close to Athens International Airport (Attica Region). The number of eggs laid in ovitraps was similar in all intervention and control plots. However, a significant reduction in egg hatch rate was recorded in the SIT plot in comparison with both control plots starting two weeks after the first release. This trial validates the logistics (transportation, releases handling and monitoring) as a major step towards implementing efficient, environmentally safe control approaches as an additional tool against the invasive Aedes species in Greece and more widely in Europe.

Critical to successful application of the sterile insect technique against Aedes albopictus (Skuse) is the development of an efficient and standardized rearing protocol to be employed in the mass production system. In this study, several life history traits of Ae. albopictus were analyzed to identify upper and lower thresholds of larval density and diet concentration. Survival to pupation, time to pupation, and sex ratio were evaluated under a range of larval densities (0.5–5 larvae/ml) and food levels (0.05–1.6 mg/larva/d) using two larval diets (one locally developed; one developed by the FAO/IAEA). The larvae reared at 28 °C, at a density of 2 larvae/ml and receiving a food dose equal to 0.6 mg/larva/d of a diet consisting of 50% tuna meal, 50% bovine liver powder (the FAO/IAEA diet), and, as an additive, 0.2 g of Vitamin Mix per 100 ml of diet solution, developed in 5 d and had 90% survival to the pupal stage. With this rearing regime male pupae production 24 h after the onset of pupation was the highest; these pupae were ∼94% male.

We explored the possibility to improve male/female separation (sexing) in Aedes albopictus by selection of two strains, one toward increasing sex dimorphism and another toward increasing protandry. In the laboratory we selected and crossed small males with large females to exploit dimorphism, and early pupating males with late pupating females to exploit protandry. While selection for enhanced dimorphism was not a profitable character, the selection for enhanced protandry up to F₁₀ produced significant improvement in the time interval between male and female pupation. By collecting the pupae at 24 h from the beginning of pupation, without any sieving operation, we obtained about 28.50% of pupae (calculated in relation to the estimated initial number of first instar larvae used), vs 26.49% we had in the control strain, and, more interestingly, when checking the sex ratio of these pupae we observed a presence of females of 0.92% vs 23.02% in the control strain. We also modified our egg hatching protocol from the previous standard procedure that required keeping the eggs in the glass hatching container overnight (for about 14-16 h) to a new protocol where eggs are kept in the hatching container for 4 h in order to obtain more synchronized larvae. This was possible without any reduction in the egg hatching rate. In Aedes albopictus it is possible to develop hyper-protandric strains useful to produce male pupae without applying other sexing systems. This represents a considerable achievement assisting the Sterile Insect Technique application, allowing improvement of the current sexing method based on mechanical separation. More investigations are under way in order to further enhance the male productivity capacity of the strain and to determine whether the selection has any impact on the male fitness parameters.

Genetic based mosquito control methods have been gaining ground in recent years for their potential to achieve effective suppression or replacement of vector populations without hampering environments or causing any public health risk. These methods require the mass rearing of the target species in large facilities sized to produce millions of sterile males, as already well established for a number of insects of agricultural importance. Assessing the performance of released males in Sterile Insect Technique (SIT) control programs is of the utmost importance for the success of the operation. Besides the negative effects of mass rearing and sterilization, the handling of sterilized insects and shipment to distant areas may also negatively impact the quality of sterilized males. The aim of the current study was to design and executive quality control (QC) tests for sterilized Aedes albopictus (Asian tiger mosquito) males delivered by air shipment from the mass production facility located in Italy to Greece and Montenegro field release sites. Mass reared mosquito strains were based on biological materials received from Italy, Greece and Montenegro. Tests conducted at the mass rearing facility before transportation revealed a rather high residual female contamination following mechanical sex separation (approximately 1.5% females, regardless of the mosquito strain). Irradiated males of all three mosquito strains induced high levels of sterility to females. Shipment lasting approximately 24 h resulted in approximately 15% mortality, while when shipment lasted nearly two days this increased to almost 40%. The flight ability of sterilized males following one day transportation time was satisfactory (over 60%). The response of sterile males to food and water starvation was comparable and slightly lower than that of wild non-transported males. Longevity of sterile males was shorter than that of wild counterparts and it seems it was not affected by mating to wild females. Both mating propensity and mating competitiveness for wild virgin females was higher for the wild, control males compared to the sterile, transported ones. Overall, the performance of sterile male Ae. albopictus delivered from the mass rearing facility of Italy to Greece in approximately 24 h was satisfactory. Transportation lasting two days or longer incurred detrimental effects on males, which called into question the outcome of the SIT release programs. In conclusion, our results demonstrate the need of quality control procedures, especially when sterile male production facilities are not near to the releasing point. Transportation could be a serious drawback for the implementation of Sterile Insect Releases and, consequently, it is important to establish an efficient and fast transportation of sterilized males in advance.