Sterile Insect Technique (SIT) field trial targeting the suppression of Aedes albopictus in Greece.

The sterile insect technique (SIT) involves releasing large numbers of sterile males to outcompete wild males in mating with females, leading to a decline in pest populations. In the current study, we conducted a suppression trial in Greece against the invasive dengue vector mosquito Aedes albopictus (Skuse) through the weekly release of sterile males for 22 weeks from June to September 2019. Our approach included the long-distance transport of sterile mosquitoes, and their release at a density of 2,547 ± 159 sterile males per hectare per week as part of an area-wide integrated pest management strategy (AW-IPM). The repeated releases of sterile males resulted in a gradual reduction in egg density, reaching 78% from mid-June to early September. This reduction remained between 70% and 78% for four weeks after the end of the releases. Additionally, in the SIT intervention area, the ovitrap index, representing the percentage of traps containing eggs, remained lower throughout the trial than in the control area. This trial represents a significant advance in the field of mosquito control, as it explores the viability and efficacy of producing and transporting sterile males from a distant facility to the release area. Our results provide valuable insights for future SIT programmes targeting Ae. Albopictus, and the methodology we employed can serve as a starting point for developing more refined and effective release protocols, including the transportation of sterile males over long distances from production units to intervention areas.

Title: Essai sur le terrain de la Technique de l'Insecte Stérile (TIS) ciblant la suppression d'Aedes albopictus en Grèce. Abstract: La technique de l'insecte stérile (TIS) consiste à libérer un grand nombre de mâles stériles pour supplanter les mâles sauvages lors de l'accouplement avec les femelles, entraînant ainsi un déclin des populations de nuisibles. Dans la présente étude, nous avons mené un essai de suppression en Grèce contre le moustique vecteur invasif de la dengue, Aedes albopictus (Skuse), par le biais de la libération hebdomadaire de mâles stériles pendant 22 semaines de juin à septembre 2019. Notre approche comprenait le transport sur de longues distances de moustiques stériles, et leur lâcher à une densité de 2 547 ± 159 mâles stériles par hectare et par semaine dans le cadre d'une stratégie de lutte intégrée contre les nuisibles à l'échelle de la zone (AW-IPM). Les lâchers répétés de mâles stériles ont entraîné une réduction progressive de la densité des œufs, atteignant 78 % de la mi-juin au début septembre. Cette réduction est restée entre 70 % et 78 % pendant quatre semaines après la fin des lâchers. De plus, dans la zone d'intervention de la TIS, l'indice d'oviposition, représentant le pourcentage de pièges contenant des œufs, est resté plus faible que dans la zone témoin tout au long de l'essai. Cet essai représente une avancée significative dans le domaine de la lutte contre les moustiques, car il explore la viabilité et l'efficacité de la production et du transport de mâles stériles depuis une installation éloignée vers la zone de lâcher. Nos résultats fournissent des informations précieuses pour les futurs programmes de TIS ciblant Ae. albopictus et la méthodologie que nous avons utilisée pourra servir de point de départ pour développer des protocoles de libération plus raffinés et plus efficaces, y compris le transport de mâles stériles sur de longues distances depuis les unités de production jusqu'aux zones d'intervention.

Correction: Aedes albopictus microbiota: Differences between wild and mass-reared immatures do not suggest negative impacts from a diet based on black soldier fly larvae and fish food.

[This corrects the article DOI: 10.1371/journal.pone.0292043.].

Effects of the Weather on the Seasonal Population Trend of Aedes albopictus (Diptera: Culicidae) in Northern Italy.

BACKGROUND: Aedes albopictus, the Asian tiger mosquito, has become a prevalent pest in Italy, causing severe nuisance and posing a threat of transmission of arboviruses introduced by infected travelers. In this study, we investigated the influence of weather parameters on the seasonal population density of Aedes albopictus. METHODS: A Bayesian approach was employed to identify the best meteorological predictors of species trend, using the eggs collected monthly from 2010 to 2022 by the Emilia-Romagna regional monitoring network. RESULTS: The findings show that the winter-spring period (January to May) plays a crucial role in the size of the first generation and seasonal development of the species. CONCLUSIONS: A temperate winter and a dry and cold March, followed by a rainy and hot spring and a rainy July, seem to favor the seasonal development of Ae. albopictus.

Effect of 2 sex-sorting time schedules on SIT facility management.

Improvements are needed in mosquito mass-rearing to effectively implement the sterile insect technique (SIT). However, managing this technique is challenging and resource intensive. SIT relies on mass rearing, sterilization, and release of adult males to reduce field populations. Maintaining an acceptable level of female presence, who can transmit viruses through biting, is crucial. Females are also essential for facility sustainability. Sex sorting plays a vital role in the production process, and our current mechanical sorting approach aims to obtain a high number of adult males with minimal female contamination within 24 h of pupation. Utilizing protandry helps control female contamination. While the 24-h sorting period achieves desired contamination levels, it may not yield enough females to sustain breeding lines, leading to increased labor costs that impact project sustainability. By delaying the sorting procedure to 48 h, we obtained sufficient females to sustain breeding lines, achieving a balance between male production and female contamination using the automatic version of the Fay-Morlan device as the sorting tool.

Two invasions at once: update on the introduction of the invasive species Aedes aegypti and Aedes albopictus in Cyprus - a call for action in Europe.

Aedes aegypti, the yellow fever mosquito and Aedes albopictus, the tiger mosquito, continue to expand their geographical distribution, reshaping the European epidemiological risks for mosquito-borne diseases. The reintroduction of Aedes aegypti near the airport and port in Larnaka and the detection of Aedes albopictus near the marina and old port of the Limassol area in Cyprus are reported herein. The measures taken to investigate these events included (i) communication to health authorities, (ii) expert on-site visits and verification of findings, (iii) enhanced active surveillance, and (iv) development of an Emergency Action Plan followed by a Contingency Plan. These emergency action plans were developed to delimitate the infested areas and to prevent the spreading of the mosquito populations into new areas. The general principles are presented along with their rationale to serve as guidelines for other geographical regions targeting suppression/eradication with a sterile insect technique component. In parallel, this manuscript serves as a call for action at the European level to impede the further spread of these species and support the activities being undertaken in Cyprus to combat the incursions of Aedes invasive species.

Title: Deux invasions à la fois : le point sur l'introduction des espèces envahissantes Aedes aegypti et Aedes albopictus à Chypre ­ un appel à l'action en Europe. Abstract: Aedes aegypti, le moustique de la fièvre jaune, et Aedes albopictus, le moustique tigre, continuent à étendre leurs aires de distribution, redessinant les risques épidémiologiques des maladies transmises par les moustiques en Europe. La réintroduction d'Aedes aegypti près de l'aéroport et du port de Larnaka, et la détection d'Aedes albopictus près de la marina et du vieux port de la zone de Limassol à Chypre sont rapportées dans cette étude. Les mesures prises pour investiguer ces évènements ont inclus : (i) la communication avec les autorités sanitaires, (ii) des visites d'experts sur site et la vérification des résultats, (iii) une surveillance active augmentée et (iv) le développement d'un Plan d'Actions d'Urgence suivi d'un Plan de Contingence. Des plans d'action d'urgence ont été développés pour délimiter les zones infestées et prévenir l'expansion des populations de moustiques à d'autres zones. Les principes généraux sont présentés, en même temps que leur justification, pour guider d'autres zones géographiques visant la suppression / éradication avec une composante de technique de l'insecte stérile. En parallèle, cet article sert d'appel, au niveau européen, à prévenir la diffusion toujours plus importante de ces espèces et pour appuyer les activités à mener à Chypre pour combattre les incursions des espèces invasives d'Aedes.

Aedes albopictus microbiota: Differences between wild and mass-reared immatures do not suggest negative impacts from a diet based on black soldier fly larvae and fish food.

The "Sterile Insect Technique" (SIT), a promising method to control Aedes albopictus, the Asian tiger mosquito, is gaining increasing interest. Recently, the role of microbiota in mosquito fitness received attention, but the link between microbiota and larval diet in mass rearing programs for SIT remains largely unexplored. We characterized the microbiota of four larval instars, pupae and eggs of non-wild (NW) lab-reared Ae. albopictus fed with a diet based on Black soldier fly (Hermetia illucens) larvae powder and fish food KOI pellets. We compared it with wild (W) field-collected individuals and the bacterial community occurring in rearing water-diet (DIET). A total of 18 bacterial classes with > 0.10% abundance were found overall in the samples, with seven classes being especially abundant. Overall, the microbiota profile significantly differed among NW, W and DIET. Verrucomicrobiae were significantly more abundant in W and DIET, Bacteroidia were more abundant in NW and DIET, and Gammaproteobacteria were only more abundant in W than in DIET. W-eggs microbiota differed from all the other groups. Large differences also appeared at the bacterial genus-level, with the abundance of 14 genera differing among groups. Three ASVs of Acinetobacter, known to have positive effects on tiger mosquitoes, were more abundant in NW than in W, while Serratia, known to have negative or neutral effects on another Aedes species, was less abundant in NW than in W. The bacterial community of W-eggs was the richest in species, while dominance and diversity did not differ among groups. Our data show that the diet based on Black soldier fly powder and fish food KOI influences the microbiota of NW tiger mosquito immature stages, but not in a way that may suggest a negative impact on their quality in SIT programs.

Monitoring of Insecticide Resistance Mutations and Pathogen Circulation in Sand Flies from Emilia-Romagna, a Leishmaniasis Endemic Region of Northern Italy.

The continuously expanding distribution of sand flies, proven vectors of Leishmania and of several phleboviruses, is a growing public health issue in Europe. Especially in Italy, visceral leishmaniasis (VL) is occurring with increasing incidence northward, in previously non-endemic provinces. Around the globe, disease elimination efforts largely focus on sand fly vector insecticidal control, often leading to the development of resistance. In Emilia-Romagna (ER), northern Italy, insecticides are heavily applied for agricultural and mosquito control, but not specifically against sand flies. Here, we investigated the sand fly species composition in certain environmental settings in ER provinces and monitored the presence of pyrethroid resistance mutations and pathogen circulation. Phlebotomus perfiliewi, a dominant vector of Leishmania infantum, was detected almost exclusively in the region. No mutations in the voltage-gated sodium channel gene, e.g., knock-down resistance mutations I1011M, L1014F/S, V1016G, or F1020S, were recorded. Pathogen monitoring revealed that almost 40% of the tested sand fly pools were positive for Leishmania, while the presence of Toscana and Fermo phleboviruses was also observed in much lower frequencies (≤3% positive pools). Regular epidemiological and entomological monitoring, alongside resistance surveillance, is highly recommended to ensure the sustainability and efficiency of vector control interventions.

Infravec2 guidelines for the design and operation of containment level 2 and 3 insectaries in Europe.

With the current expansion of vector-based research and an increasing number of facilities rearing arthropod vectors and infecting them with pathogens, common measures for containment of arthropods as well as manipulation of pathogens are becoming essential for the design and running of such research facilities to ensure safe work and reproducibility, without compromising experimental feasibility. These guidelines and comments were written by experts of the Infravec2 consortium, a Horizon 2020-funded consortium integrating the most sophisticated European infrastructures for research on arthropod vectors of human and animal diseases. They reflect current good practice across European laboratories with experience of safely handling different mosquito species and the pathogens they transmit. As such, they provide experience-based advice to assess and manage the risks to work safely with mosquitoes and the pathogens they transmit. This document can also form the basis for research with other arthropods, for example, midges, ticks or sandflies, with some modification to reflect specific requirements.

Cuticle Modifications and Over-Expression of the Chitin-Synthase Gene in Diflubenzuron-Resistant Phenotype.

Insecticide resistance is a major threat challenging the control of harmful insect species. The study of resistant phenotypes is, therefore, pivotal to understand molecular mechanisms underpinning insecticide resistance and plan effective control and resistance management strategies. Here, we further analysed the diflubenzuron (DFB)-resistant phenotype due to the point-mutation I1043M in the chitin-synthase 1 gene (chs1) in the mosquito Culex pipiens. By comparing susceptible and resistant strains of Cx. pipiens through DFB bioassays, molecular analyses and scanning electron microscopy, we showed that the I1043M-resistant mosquitoes have: (i) a striking level of DFB resistance (i.e., resistance ratio: 9006); (ii) a constitutive 11-fold over-expression of the chs1 gene; (iii) enhanced cuticle thickness and cuticular chitin content. Culex pipiens is one of the most important vector species in Europe and the rapid spread of DFB resistance can threaten its control. Our results, by adding new data about the DFB-resistant phenotype, provide important information for the control and management of insecticide resistance.

Efficacy Evaluation of Oregano Essential Oil Mixed with Bacillus thuringiensis israelensis and Diflubenzuron against Culex pipiens and Aedes albopictus in Road Drains of Italy.

Mosquito management programs in the urban environment of Italian cities mainly rely on larval control with conventional insecticides, primarily targeting the road drains that constitute the principal mosquito breeding sites encountered in public. The repeated utilization of synthetic insecticides may have adverse effects on non-targets and lead to resistance development issues, while the performance of biopesticides encounters limitations in field use. Botanical insecticides as single larval control agents or in binary mixtures with conventional insecticides have been extensively studied in the laboratory as an effective and eco-friendly alternative mosquito control method with promising results. The study herein concerns the investigation, for the first time under realistic conditions in the field, of the joint action of a carvacrol-rich oregano Essential Oil (EO) with two conventional insecticides, namely, the insect growth regulator diflubenzuron and the bio-insecticide Bacillus thuringiensis israelensis (B.t.i.), in road drains in Crevalcore city, Italy, against Culex pipiens and Aedes albopictus. According to the obtained results, the application of both plain EO and its mixtures with diflubenzuron and B.t.i. exerted very high efficacy in terms of immature mosquito population reduction over a two-week period. Three weeks after treatment, the performance of the oil and its mixtures diminished but remained high, while the addition of diflubenzuron potentiated the persistent action of the oil against Cx. pipiens. These findings are indicative of the potential of mixing carvacrol-rich EO with diflubenzuron and B.t.i. as an efficient eco-friendly alternative to mono-insecticide applications in road drains against Cx. pipiens and Ae. albopictus larvae.

Aedes albopictus Sterile Male Production: Influence of Strains, Larval Diet and Mechanical Sexing Tools.

The sterile insect technique (SIT) is a biologically based method of pest control, which relies on the mass production, sterilization, and release of sterile males of the target species. Since females can transmit viruses, it is important to develop a mass rearing system to produce a large number of males with a low presence of females. We evaluated the effects of different strains, larval diets and sexing tools on male productivity and residual female presence for the application of SIT against Aedes albopictus. Strains coming from Italy, Germany, Greece, and Montenegro, with different levels of colonization, were reared with three larval diets: IAEA-BY, BLP-B and SLP-BY. Developed pupae were sexed using two different mechanical methods: sieve or Fay-Morlan separator. The results proved that adoption of the Fay-Morlan separator increased the productivity and limited the female presence. The IAEA-BY diet showed the lowest female contamination. Strains with a high number of breeding generations showed a decreased productivity and an increased female presence. Increased female presence was found only in extensively reared strains and only when the sorting operation was conducted with sieves. We hypothesize that extensive colonization may determine a size reduction which limits the sexing tool efficiency itself.

Arbovirus Screening in Mosquitoes in Emilia-Romagna (Italy, 2021) and Isolation of Tahyna Virus.

Several viruses can be transmitted by mosquitoes. We searched some of these viruses in 20,778 mosquitoes, collected in 95 traps on the plains of Emilia-Romagna (North of Italy) in 2021. We detected West Nile virus (WNV) and Usutu virus (USUV) in pools of Culex (Cx.) pipiens. In addition, we detected two insect-specific flaviviruses in three pools of Aedes (Ae.) caspius and in two of Ae. vexans. Tahyna virus (TAHV) was detected in six pools, three of Ae. caspius and three of Cx. pipiens, and one isolated strain was obtained from one of the Ae. caspius pools. Moreover, we detected TAHV in pools of several mosquito species (Ae. caspius, Ae. vexans, Ae. albopictus, Anopheles maculipennis s.l.) collected in the previous year of surveillance. Our data indicate Ae. caspius as the species most infected with TAHV in the surveyed area. Together with the likely plasticity of the cycle, we reported strong genome stability of the TAHV, probably linked to a successful adaptation of the virus to its ecological niche. Interestingly, in six pools of Cx. pipiens we detected two associated viruses among USUV, WNV, TAHV and all the three viruses in two pools. This result allows us to assume the presence of particular conditions that prompt the circulation of arboviruses, creating the conditions for viral hot spots. While no human diseases related to Tahyna virus were reported in Italy, its detection over the years suggests that it is worth investigating this virus as a potential cause of disease in humans in order to assess its health burden. IMPORTANCE We reported in this work the detection of three Arboviruses (Arthropod-borne viruses) in mosquitoes collected in Emilia-Romagna in 2021. In addition to West Nile and Usutu viruses, which were reported from more than 10 years in the study area, we detected and isolated Tahyna virus (TAHV). We also reported detections of TAHV obtained in previous years of surveillance in different species of mosquitoes. TAHV is the potential causative agent of summer influenza-like diseases and also of meningitis. Even if human cases of disease referable to this virus are not reported in Italy, its relevant presence in mosquitoes suggests investigating the possibility they could.

A Mark-Release-Recapture Study to Estimate Field Performance of Imported Radio-Sterilized Male Aedes albopictus in Albania.

The pathogen transmitting Aedes albopictus mosquito is spreading rapidly in Europe, putting millions of humans and animals at risk. This species is well-established in Albania since its first detection in 1979. The sterile insect technique (SIT) is increasingly gaining momentum worldwide as a component of area-wide-integrated pest management. However, estimating how the sterile males will perform in the field and the size of target populations is crucial for better decision-making, designing and elaborating appropriate SIT pilot trials, and subsequent large-scale release strategies. A mark-release-recapture (MRR) experiment was carried out in Albania within a highly urbanized area in the city of Tirana. The radio-sterilized adults of Ae. albopictus Albania strain males were transported by plane from Centro Agricoltura Ambiente (CAA) mass-production facility (Bologna, Italy), where they were reared. In Albania, sterile males were sugar-fed, marked with fluorescent powder, and released. The aim of this study was to estimate, under field conditions, their dispersal capacity, probability of daily survival and competitiveness, and the size of the target population. In addition, two adult mosquito collection methods were also evaluated: BG-Sentinel traps baited with BG-Lure and CO2, (BGS) versus human landing catch (HLC). The overall recapture rates did not differ significantly between the two methods (2.36% and 1.57% of the total male released were recaptured respectively by BGS and HLC), suggesting a similar trapping efficiency under these conditions. Sterile males traveled a mean distance of 93.85 ± 42.58 m and dispersed up to 258 m. Moreover, they were observed living in the field up to 15 days after release with an average life expectancy of 4.26 ± 0.80 days. Whether mosquitoes were marked with green, blue, yellow, or pink, released at 3.00 p.m. or 6.00 p.m., there was no significant difference in the recapture, dispersal, and survival rates in the field. The Fried competitiveness index was estimated at 0.28. This mark-release-recapture study provided important data for better decision-making and planning before moving to pilot SIT trials in Albania. Moreover, it also showed that both BG-traps and HLC were successful in monitoring adult mosquitoes and provided similar estimations of the main entomological parameters needed.

Field Performance Assessment of Irradiated Aedes albopictus Males Through Mark-Release-Recapture Trials With Multiple Release Points.

Mark-release-recapture (MRR) trials have been conducted in Northern Italy to evaluate the capacity of radio-substerilized Aedes albopictus males to survive, disperse, and engage in mating in the field. Two MRR sessions with the human landing collection method (HLC) were conducted with the simultaneous release of irradiated males marked with four different pigment colors. The survival and dispersal rates seem to be influenced more by environmental factors such as barriers, shading, and vegetation rather than weather parameters. In this study, we confirmed a positive linear relationship between the sterile adult male's daily survival rate and the relative humidity previously reported in similar experimental conditions and a different dispersal capacity of the released A. albopictus males in low- (NDVI index <0.4) and high (NDVI index >0.4)-vegetated areas. Consistent with previous studies, A. albopictus males have their maximal dispersion in the first days after release, while in the following days the males become more stationary. The similar field performances obtained with marked and unmarked radio-sterilized and untreated A. albopictus males on similar environments confirm the negligible effects of irradiation and marking procedures on the quality of the males released. The similar sterile to wild (S/W) male ratio measured in high- and low-vegetation areas in the release sites indicates a similar distribution pattern for the wild and the released sterile males. According to the MRR data collected, the Lincoln index estimated different A. albopictus mean population densities in the study areas equal to 7,000 and 3,000 male/ha, respectively.

Optimization of Aedes albopictus (Diptera: Culicidae) Mass Rearing through Cost-Effective Larval Feeding.

Aedes (Stegomyia) albopictus (Skuse, 1895) is an invasive important medical and veterinary pest species. The sterile insect technique (SIT) involves the mass rearing of males, and their sterilization and release into the habitat to compete with wild males. Our research objective was to compare the effectiveness of three larval diet recipes (IAEA-BY, BCWPRL, and MIX-14) in the laboratory rearing of Ae. albopictus males to evaluate the available economical feeding alternatives. The separation of sexes was done in the pupal stage by sieving. Reared males were tested for flight capacity and longevity. The application of the BCWPRL diet resulted in a higher portion of sieved male pupae than females, but the development of males was the slowest, and the number of obtained males (pupae and adults) was lower compared to the other two diets. The adult mean survival time was the highest in males fed with MIX-14 and the lowest in males fed with IAEA-BY. Males fed by IAEA-BY also demonstrated higher initial mortality in the adult stage. The diets BCWPRL and MIX-14 are economically more convenient than IAEA-BY (2.28 and 5.30 times cheaper, respectively). The cheapest diet, MIX-14, might represent a candidate for replacing the effective but still expensive IAEA-BY larval diet, providing lower costs of sterile male production.

First evidence of pyrethroid resistance in Italian populations of West Nile virus vector Culex pipiens.

Culex pipiens (Linnaeus), one of the most abundant mosquito species in Europe, plays a crucial role in the endemic transmission of West Nile virus and caused the large outbreak with >1600 human cases in 2018. Although evidence of resistance to pyrethroids has been reported for Cx. pipiens populations from Spain and Greece, resistance monitoring has been largely neglected in Italy. Herein, we investigate susceptibility of Italian Cx. pipiens populations to the pyrethroids permethrin and deltamethrin. Results from WHO-tube-bioassays revealed mortalities ranging from 14-54%, indicating high levels of resistance, in four out of 10 populations exposed to permethrin (0.75%) and of 63% in one of three populations exposed to deltamethrin (0.05%). Reduced susceptibility (mortality<98%) was detected in almost all other populations. A clear association is shown between the resistant phenotype and the presence of kdr-alleles in position 1014 of the VSSC, strongly suggesting its role in reducing susceptibility. The study provides the first evidence of pyrethroid-resistance in Italian Cx. pipiens populations and reports levels of resistance paralleled in the European region only in Turkey. This highlights the urgent need to implement insecticide-resistance management plans to restore the efficacy of the nowadays only chemical weapon available to control arbovirus transmission in Europe.

Quality Control Methods for Aedes albopictus Sterile Male Transportation.

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.

Integrated control of Aedes albopictus in Southwest Germany supported by the Sterile Insect Technique.

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.

Historical samples reveal a combined role of agriculture and public-health applications in vector resistance to insecticides.

BACKGROUND: Insecticide resistance is the major threat to vector control and for the prevention of vector-borne diseases. Because almost all insecticides used against insect vectors are or have been used in agriculture, a connection between agricultural insecticide use and resistance in insect vectors has been hypothesized. However, it is challenging to find a causal link between past agricultural use of insecticides and current resistance in vector populations without historical data series. Here we investigated the relative contribution across time of agricultural and public-health insecticide applications in selecting for diflubenzuron (DFB) resistance in Culex pipiens populations. Using DNA sequencing, we looked for DFB resistant mutations in current and historical mosquito samples, dating back to the 1980s-1990s, when DFB was used in agriculture but not yet in mosquito control. RESULTS: In the samples collected before the introduction of DFB in vector control, we found the resistant mutation I1043M in rural regions but not any of the neighboring urban and natural areas, indicating that the selection pressure was derived by agriculture. However, after the introduction of DFB for vector control, the resistant mutations were found across all study areas showing that the initial selection from agriculture was further boosted by the selection pressure imposed by the mosquito control applications in the 2000s. CONCLUSIONS: Our findings support a combined role of agricultural and public-health use of insecticides in vector resistance across time and call for specific actions in integrated resistance management, including increased communication between agriculture and health practitioners. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.