Safety, regulatory and environmental issues with sterile insect technique-based mosquito vector control in European countries

The globalisation of trade is opening the way to the spread of species in new regions where they may cause negative impacts. Invasive mosquito species such as Aedes aegypti and Aedes albopictus are raising concern for their capacity to transmit several arboviruses of public health and veterinary importance. Currently available integrated vector management measures do not achieve satisfactory results when deployed against these urban mosquitoes. Moreover, insecticides are losing their effectiveness owing to the resistance developed by the target species. Policies regulating the use of insecticides are progressively restricting their market availability and this trend is expected to continue. Genetic control methods, such as the sterile insect technique (SIT), based on the use of irradiation to sterilise male mosquitoes, are showing good efficacy in pilot trials at local scales in some Aedes albopictus-colonised urban areas in Europe, without any negative effects. The main limitation is the cost, which may be significantly reduced through the introduction of automation in the mass rearing and drone technology in the field release. These technological advancements require substantial investments at a scale that can only be achieved with centralised production and extensive distribution, which in turn may be granted only if the authorisation frameworks, including the regulation of international transportation and aerial release in an urban setting, are clarified and matured.

section_content: The human capacity to deal with mosquito-borne diseases (MBDs), such as malaria, dengue, lymphatic filariasis, chikungunya, Zika, yellow fever, Japanese encephalitis, West Nile fever and many others, leading to millions of cases every year, is largely based on the deployment of integrated vector management (IVM), including the use of insecticides, source removal, community participation and repellents protecting the hosts [1, 2]. Mosquito species respond through evolutionary processes to the pressure exerted by insecticides, with increasing evidence of resistance. The phenomenon of resistance to insecticides was observed in several different ecological settings when the use of an insecticide was repeated multiple times over large areas [3]. The only case where no evidence of resistance was reported following many years of continuous treatment of mosquitoes may be that of Bacillus thuringiensis israelensis, a microbial insecticide containing at least four toxic proteins with specific modes of action [4]. Together with resistance, insecticides show other unintentional negative effects, including the impact on non-target organisms, contamination of the ecosystem and sublethal toxicity to higher animals. The increasing evidence of the negative environmental effects of insecticides induced the authorities to develop stringent policies to regulate their marketing and use. In this regard, in the European Union (EU), EU Regulation No. 528/2012 is considered to be at the forefront [5]. 

The public in western countries is also developing a negative perception of insecticides, with many homeowners forbidding the local administration to spray insecticides on their properties [6]. In countries where MBDs are endemic, the opposition to insecticide use is much less pronounced https://doi. org/10. 20506/rst. 41. 1. 3314 and safe, confined conditions. In this regard, Aedes mosquito species of public health importance, such as Ae. aegypti and Ae. albopictus, seem to be intrinsically more prone to mate in cages than most of the Anopheles mosquito species, owing to their specific mating behaviour [17, 18]. Other more dispersive Aedes species, targeted in Europe for their nuisance value, such as Ae. vexans and Ae. sticticus, are more reluctant to colonise in factory production systems [19]. 

The phenomenon of invasion of new areas by Aedes species, while not a recent development [20], is currently raising alarm because of its worldwide scale and surprising rapidity. This puts pressure on the relevant authorities to organise adequate preventive measures [21, 22]. Invasive Aedes species such as Ae. aegypti and Ae. albopictus should be regarded as particularly suitable for SIT application for several reasons:

-they are relatively easy to mass rear -their suppression or even elimination from the newly colonised areas poses no threat to local biodiversity, because they do not belong to the local fauna -both these species flourish in urban environments, where contact with humans is frequent and where their limited active dispersal capacity makes it possible to apply SIT -finally, the current IVM measures do not guarantee a satisfactory control level, and dengue, chikungunya and Zika virus epidemics are on the increase in many regions. 

Along with the negative impact of globalisation on biocenosis and human well-being, it is possible to envisage a positive side effect: the spread of tropical vector species into more developed temperate countries is mobilising energy, research activities and, more importantly, investment in technology. The rising threat has focused attention on promoting new understanding and technological solutions which are contributing to the advancement in mosquito genetic control worldwide [23]. With the help of new technological tools and approaches, researchers are now in a solid position to overcome the two main obstacles to the broad application of SIT and other genetic-based control methods against mosquito vectors: achieving efficient production of large quantities of sterile males at a competitive cost and developing field release methods that ensure the homogeneous distribution of sterile male mosquitoes in urban areas.
section_name: Introduction

section_content: Live adult mosquitoes are very delicate, therefore delivery time must be reduced to a minimum, which implies reliance on air-shipping companies with express service [24]. These companies recognise the difficulties in managing the transport of live mosquitoes and usually do not provide any guarantee for the quality and timing of a delivery. 

because of the different balance of cost and benefit, but it may increase in the near future as public awareness and environmental expectations grow. Meanwhile, the number of active ingredients available for mosquito control is rapidly declining on a global scale. The trend is expected to continue with the removal from the market of products showing evidence of negative environmental impact. It becomes more and more difficult for the pesticide industries to develop new products which comply with the current regulations. The research community is, therefore, strongly engaged in finding new tools and methods to be deployed in the endless struggle against mosquitoes and MBDs. Such innovations must be able to avoid natural development of resistance and to be highly effective against the target species, without negative repercussions for wildlife and human health. 

Among the proposals for additions to IVM that have appeared in recent years is the sterile insect technique (SIT). This approach is long established in the control of agricultural and livestock pest insects, dating back to the 1950s when it was first successfully applied for the suppression of Cochliomyia hominivorax in the South-east United States of America (USA), before expansion of the programme throughout the USA, Mexico and Central America [7, 8]. After that success, SIT was applied against several agricultural pest species in different ecological and climatic conditions [9, 10]. A complete repertoire of the SIT programmes attempted and those currently running is reported in the International Database on Insect Disinfestation and Sterilization (IDIDAS) [11]. The sterile insect control system has now been extended to mosquito control through advances in mass rearing and delivery techniques that make operational implementation more practical [12, 13, 14, 15].
section_name: Challenges in area-wide SIT deployment against mosquitoes

section_content: While SIT is applied operationally on an area-wide scale against several Tephritid fruit fly species, codling moths, screwworm flies and tsetse flies, it is still at the pilot scale on mosquitoes. 

The historical reasons behind this apparently difficult to understand situation have been well analysed by Benedict [16]. 

One main point is that, while IVM may affect more than one mosquito species at a time in cases where they are sympatric, by design SIT controls only one species or mating complex. Unless the mosquito mass-rearing facility is organised to produce and distribute more than one species, the entire campaign affects only the target species. This point is not trivial, because in many situations multiple species are involved in disease transmission and should be targeted at the same time. 

Another basic assumption allowing effective application of genetic control is that the target species should be easily colonised in artificial environments and mass reared in efficient
section_name: The SIT application against mosquitoes

section_content: In general, sterile insects can be classified as beneficial organisms, as reported by the International Plant Protection Convention (IPPC) in its International Standard for Phytosanitary Measures (ISPM) No. 3, 'Guidelines for the export, shipment, import and release of biological control agents and other beneficial organisms' [28]. This document is mainly designed for species of agricultural importance, however, and the conditions for extrapolating it to species of health importance should be better clarified and explained [29]. 

The potential risks related to the intentional/unintentional release of sub-sterile male mosquitoes or residual females together with the sterile males are specific to public health and should be evaluated within that regulatory perspective. 

The current national authorisation procedure to import sterile male mosquitoes differs from country to country, even within the EU. Sometimes the regulatory pathway is unclear, most likely due to the novelty of the case for the relevant authorities. Sometimes it is even difficult to find out which institution has the specific responsibility and authority to address the issue. The author's experience in local urban SIT pilot applications in Italy and several nearby countries has involved both national (environmental and health departments) and local authorities (city and regional governments) providing the authorisations needed for delivery and release of sterile mosquitoes. For example, in the case of Montenegro, a one-year temporary permit was released by the Agency for Environmental Protection; in the case of Albania, the authorisation was provided by the National Institute of Public Health; in the case of Germany, the permit was delivered by the state of Baden-Württemberg where the releases were planned; while, in the case of Greece, an informal authorisation was obtained through a large stakeholder meeting including the Benaki Phytopathological Institute, the Greek Atomic Energy Commission, the University of Thessaly, the Municipality of Markopoulo, the Prefecture of Attica, the Ministry of Health, the Ministry of Rural Development and Food, and the Greek Pest Control Association. 

Table I shows recent experiences of moving sterile male mosquitoes across borders for release in the European scenario. Even the authorisation for conducting pilot feasibility studies may be time consuming and difficult to achieve [30]. Consequently, sufficient harmonisation of legislation allowing reasonable investment for this approach to vector control has not yet occurred in Europe. 

Risk analysis specifically related to international shipments of sterile mosquitoes should be conducted within the context of the overall risk analysis of the sterile insect trade, defining the pertinent risk categories and levels (see also Enkerlin and Pereira and Quinlan et al. in this issue [29, 31] ). 

One question which inevitably arises is: what is the competitive cost for sterile males? Of course, the calculation should include the cost of production and quality control, the cost of delivery from the facility to the target area and the cost of distribution and possibly monitoring after release. The overall cost must compete favourably with the current costs of other components of IVM in similar local contexts, taking into consideration their relative effectiveness in population suppression as well as the financial capacity of entities responsible for funding local control. This is a matter for specialists in cost-effectiveness and cost-benefit analyses [25, 26, 27]. 

By looking to the mature experience of SIT application in agriculture, the basic difference observed by the author is that the crop production business has an annual budget made available by the marketing of products, while in mosquito control there is no income, just the spending capacity of the responsible public administrations that protect public health. A possible methodology assisting the cost-effectiveness analysis in the case of vector-borne diseases has been proposed by the World Health Organization (WHO) [25]. The relatively small SIT applications in urban settings do not achieve the economy of scale to allow efficient local production. This key problem can be overcome by more centralised production systems that distribute sterile males within their region, leading to a demand for international shipments in areas such as Southeast Europe or South-East Asia, where there are many small countries. In this context, clearer regulations dealing with the international trade of sterile mosquitoes will play a useful role in allowing more sustainable operation of SIT facilities. 

There is promise for the application of automation technologies, as largely developed in the industrial and logistical sectors, to the rearing of delicate creatures including mosquitoes. Close collaboration among informatics and electronics experts, mechanical engineers, production flow specialists and entomologists should be established, and a shared language developed among disciplines to achieve this ambitious goal. The challenge remains, however, that to be sustainable such high-tech facilities will require significant investment and then must rely on local-scale commitments exposed to the risk of intermittent contracts. Therefore, the solution may be a large production facility in a regional market (possibly producing multiple species or strains of mosquitoes for different local markets) with an efficient distribution network. The potential for success of this approach, however, is subject to further improvements in production processes, which are far from being accomplished in the case of mosquito species at present, plus a clear international authorisation path and national or regional regulations that support both cross-border trade and suitable delivery technologies in the control areas. 

This will help responsible international authorities to develop guidance assisting the states to adopt more harmonised legislation, including clarification of the conditions to be met by entities applying to carry out SIT. 

Exotic invasive species may be targeted (and thus sterile ones transported) because of the public health importance of mosquito vector species, thereby introducing nuanced regulatory issues. Transport entities cannot attest to the residual presence of biting females or the males not being fully sterile, two specific issues which must be considered, evaluated and defined in terms of acceptable thresholds. In the case of Ae. albopictus in Europe, all the countries involved in SIT field pilot trials decided to use their own field-collected seed material to start the production colony in Italy, to prevent the introduction of non-local genotypes. These issues need to be considered and harmonised where possible. 

Regarding the field release technology for sterile males, two main issues should be considered: the methodology and the timing of release, which affect the costs and the homogeneity of coverage. The release technology is particularly important in an urban setting where artificial barriers act as insurmountable obstacles to the active dispersal of sterile males. From the first evidence [32], drones may be well suited to reducing the costs of release and improving the coverage of the target area with the desired dose of sterile males. Conditions for the use of drones over urban areas are particularly stringent, for obvious safety reasons, and specific authorisation must be obtained for the defined target area. Common release systems should be used in multiple locations to gain additional efficiency in release operations, which would also improve distribution efficiency if the same packaging systems were used for all or most deliveries. 

The release mechanism for sterile mosquitoes may involve specific packaging requirements, such as canisters for drones. To ensure rapid and efficient delivery from a central production facility to a field release point, this final release packaging may need to be filled at the production site to avoid the need for additional handling for release, thus affecting the nature of the product being delivered by shipping carriers. This overall system requirement for efficient production and delivery is determined by the model of centralised production for economies of scale and reduced handling for automated aerial release. 

A regional factory must be able to meet regulatory conditions for multiple users and make reliable shipments to ensure regular effective releases at a range of destinations. For example, if marking sterile mosquitoes is required to make them easily distinguishable from wild ones, the currently available marking technologies, mainly based on adding substances, may have a negative impact on the quality of the males being shipped. In fact, to the author's knowledge, there is no simple method to genotype an insect once it has been made sterile by irradiation. All of these factors interact with regulatory frameworks that generally are not yet mature or harmonised.
section_name: Regulatory issues

section_content: When considering the importance of improved guidance and a clear regulatory pathway for live insect trade, one case that may be useful to examine is the possible establishment of Ae. aegypti in southern Europe. The current climatic conditions are considered suitable for the vector's establishment and this suitability is expected to increase due to ongoing climate change [33]. Moreover, because the species is already well established in a large area bordering the Black Sea, the risk of introduction into the Mediterranean Basin is real [34]. Would it be possible and convenient to build a preparedness plan with the explicit aim of early detection and prompt elimination of the species in case of introduction? Some good examples of successful SIT deployment to eliminate newly introduced species are available to guide such a plan [35, 36].
section_name: The special case of Aedes aegypti in Europe

section_content: While the effectiveness of SIT in urban Aedes control has been demonstrated in several settings, there remains the need to make the whole process involving the international framework for trade of sterile male mosquitoes more transparent and widely adopted. This requires agreement among the authorities involved. Consistency and transparency in guidance will have a strong positive effect on the applicability of SIT to mosquito control, coupled with sound technological development. Thanks to the recent advances in technology, SIT and other possible genetic control methods offer a concrete possibility to open a new era in the struggle against mosquito vectors and the diseases they transmitan era promising to suppress mosquitoes without harming other organisms. The international authorities responsible for defining the regulatory framework inside which these technologies may operate must pay attention and coordinate their efforts for the benefits of SIT to be realised in urban settings. pictus, que suscitan inquietud por su capacidad de transmitir varios arbovirus de importancia para la salud pública y veterinaria. Las medidas de lucha integrada contra vectores que existen actualmente no ofrecen resultados satisfactorios contra estos mosquitos urbanos. Además, los insecticidas están perdiendo eficacia debido a las resistencias adquiridas por las especies destinatarias. Las políticas que regulan el uso de insecticidas están restringiendo progresivamente su disponibilidad comercial, tendencia que presumiblemente va a perdurar. La aplicación experimental a escala local de métodos de control genético como la técnica del insecto estéril (sterile insect technique, SIT), basada en el uso de radiación para esterilizar a mosquitos macho, está resultando eficaz en ciertas zonas urbanas de Europa colonizadas por Aedes albopictus, sin que se observe ningún efecto negativo. La principal limitación es el costo, que cabe reducir sustancialmente introduciendo la automatización en la cría masiva y empleando drones para las sueltas sobre el terreno. Estos avances tecnológicos exigen inversiones cuantiosas, de tal magnitud que solo son factibles si se plantea una producción centralizada y una distribución a gran escala, procesos que a su vez solo serán aprobados si previamente se aclaran y perfeccionan los regímenes de autorización, incluida la regulación del transporte internacional y de la suelta aérea en medios urbanos.
section_name: Conclusions

section_name: R. Bellini

section_content: Aedes aegypti -Aedes albopictus -Control genético de mosquitos -Esterilidad sexual de insectos -Suelta desde drones -Técnicas de cría en masa. 

Cuestiones de seguridad, reglamentarias y ambientales que plantea la técnica del insecto estéril como método de control de mosquitos vectores en los países europeos mais celui-ci peut être significativement réduit en automatisant les procédures d'élevage de masse et en utilisant des drones pour les lâchers sur le terrain. Ces avancées technologiques nécessitent d'importants investissements à très grande échelle, qui ne sont réalisables qu'en centralisant la production et en élargissant la distribution ; à son tour, cette perspective ne peut être garantie qu'à travers une clarification et rationalisation des cadres réglementaires régissant les autorisations nécessaires, en particulier concernant les transports internationaux et les lâchers aériens au-dessus des zones urbaines.
section_name: Palabras clave

section_content: Aedes aegypti -Aedes albopictus -Lâcher par drone -Méthode génétique de contrôle des moustiques -Stérilisation sexuelle d'insectes -Technologie d'élevage de masse.
section_name: Mots-clés

section_name: Acknowledgements

section_content: The article was prepared in the framework of the European Union's Horizon 2020 research and innovation infrastructure project Infravec2 -Research infrastructures for the control of insect vector borne diseases under Grant Agreement No. 731060 ( 2017-2021 ).
section_name: Acknowledgements