Different bees as vectors for entomovectoring with enhanced pollination and crop protection control: current practices, use-cases and critical view on transport

and of a large scale. A novel approach developed to use bees as vectors of microbial agents, by inoculating the surface of the pollinators using dispensers in modified hives. This innovation extends the market for these products and results in better yields. A successful entomovector system requires selection of the vector pollinator most appropriate for the crop and location, based on various criteria, in combination with a registered microbial agent. Currently, pollinators and microbial agents are packed separately and combined at the point of use. Local sourcing of the pollinator in the system reduces the need for long-distance shipping of these live insects and may improve efficiency due to local adaptation but will delay use and benefits of the system research at each site/country the conducted in a In the meantime, for innovative systems employing live insects


Introduction
Both pollinators and biocontrol agents (BCAs) are broadly commercialized internationally. In 2015, according to Van Lenteren et al. (1), the combined global area treated with invertebrate and microbial agents amounted to more than 30 million hectares. Commercial pollination by bumblebees alone was already estimated to be a € 55 million industry in 2004 with over a million colonies produced worldwide (2). In order to further optimize both international functions, pollination and application of microbial BCAs were combined through entomovectoring.
The entomovectoring system is a good example of man copying nature.
It has been established for a long time that insects such as bees are able to transfer pollen and other microparticles such as bacterial cells and fungal spores from flower to flower, as demonstrated for Erwinia amylovora (3). Man has adopted this concept and used it for commercial pollination and, more recently, for the transport of microbial BCAs. Hokkanen and Menzler-Hokkanen (4) were the first to introduce the technical term 'entomovector technology' meaning the use of vectors such as insects for transporting microbial BCAs for plant protection. The entomovectoring system has the potential to increase the market further, but it also introduces new concepts that need to be understood when applying guidance on international risk assessment and shipping. For example, during transport, the vectors are packed separately from the BCA that must be applied via the dispenser when the beehive is installed in the greenhouse or on the field. The shipment itself does not differ or require extra management compared to standard pollinator transport. Misperception about the purpose and operation might occur during shipping, however, which is why detailed descriptions are necessary.
41_1_09_Temmermans & Smagge -pre-print 3/24 Already in 1992, the honeybee, Apis mellifera, was used to transfer the fungus Gliocladium roseum, a commercially available microbial BCA, for the protection of strawberry plants against the notorious plant pathogen Botrytis cinerea (5).
The entomovectoring system makes use of a unique combination to provide pollination and a protective service at the same time, by using pollinators such as the honeybee for the transport of microbial BCAs. As a result, the grower benefits from increased yields due to an increased seed set and protection against plant pathogens. There are other benefits from use of the entomovectoring system instead of spraying chemical control agents. Aside from avoiding chemicals that can have adverse effects on the environment or human health (6), the entomovectoring system can reduce water and electricity requirements for glasshouse production, resulting in a lower carbon footprint; this also fits within the framework of the European Green Deal (https://ec.europa.eu/info/strategy/priorities-2019-2024/europeangreen-deal_en) (7).
Entomovectoring reduces costs and labour hours and decreases nontarget organism exposure (8). In terms of hive management, the greatest requirement, apart from installation, is to refill the dispenser with BCA product, which takes approximately 15-30 seconds per hive, either twice a week for bumblebees or every 10 days for honeybees. Commercially available colonies require placement on a small stand, protection from rain (most of the hives have a paper-based coating) and access to pollen and nectar. Honeybees require year around maintenance to be applicable at the desired period with microbial BCAs. In addition, using pollinating insects for the delivery of the microbial BCAs assures that they will be disseminated directly onto the target location, that being the flowers from the moment they open (9). This is important as these are often the target organs for harmful plant pathogens (10,11). Furthermore, less microbial BCA is wasted because during spraying a significant part of the product ends up on the ground. This review will provide a brief recap on how entomovectoring works, especially in relation to transport. Some historic cases are presented on the need to use local species in entomovectoring to protect pollinator biodiversity. Overall, this article aims to share information with stakeholders on the new concept of entomovectoring, its advantages and disadvantages, together with a critical view on transport of pollinators for the purpose of entomovector systems, demonstrating the need for clarity around any related regulations on export and import.

Principles of entomovectoring
Entomovectoring is an interplay of several parts (Fig. 1). A certain pollinator, referred to as the vector, must deliver a microbial BCA product to the crop under protection in a way that is safe for man, the vector, and the environment. To load the vector with a sufficient amount of colony forming units (CFU) of the microbial BCA, the vector must be loaded using a dispenser. For this, the vector walks through a device filled with the BCA formulation. The dispenser must provide a way to load the vector safely and sufficiently without altering the physiological behaviour of the vector too much and without compromising its health and safety (7). To ensure that the BCA has no adverse effects on humans, vectors, or the environment, risk assessments must be completed covering safety and product registration must be in place (13).

The vector
Proper application of the microbial BCA to the flowers has a significant contribution to the successful operation of the entomovectoring system (11). Matching the appropriate vector with the plant under protection is hence of high importance. The choice for the most suitable vector is dependent on several factors: the crop species, the crop visitation rate of the vector, the flying conditions for the vector, and the vector's intrinsic capacity to disseminate microbial BCAs all impact the entomovectoring success (11).
The number of insect species that have already been used in the entomovectoring system is rather limited. The bottleneck is linking the commercial availability of the pollinator with the blooming period of the plant under protection as the pollinator life cycle is not always related to its blooming period (11). This is also why honeybees and bumblebees, two groups that are commercially available throughout the year, are very much in demand for use in the entomovectoring system, and have been the vector in many scientific reports investigating entomovectoring (13). For instance, Apis and Bombus workers differ in pollen deposition and removal as a result of different foraging behavior.
Examples of crop-pollinator links were found for numerous species of the Asteraceae family that proved to be particularly attractive to honeybees and wild bees (15). Members of the Brassicaceae family were mostly disliked by solitary bees and bumblebees, but visited frequently by honeybees. Bumblebees were notably present on plants of the Leguminosae family (15). Apart from honeybees and bumblebees, solitary bees such as Megachile rotundata, Osmia bicornis, Osmia cornuta and Osmia lignaria have been used as vectors as well (16). Plans are being made for the use of stingless bees (Meliponini) for entomovectoring in Latin America. The stingless bee species Nannotrigona perilampoides has pollinated tomatoes successfully (17). However, more research and new multiplication methods are needed for many stingless bee species before they could be used commercially (2).
The conditions under which the vectors must fly are very decisive for determining which vector would generate the best result for pollination. This is not different for the choice between bumblebees and honeybees. For example, it is known that honeybee workers do not fly, or only poorly, under rainy conditions and that rain affects their ability to disseminate BCAs (20,11). Bumblebees that are described as badweather foragers (20) perform better under rainy conditions. In addition, honeybees have been described as less suitable for entomovectoring in greenhouse crops (21). However, bumblebees can tolerate higher temperature extremes better and are also more resistant to temperature fluctuations (20). This is why bumblebees are the most used pollinators inside greenhouses (22). Nevertheless, bumblebees are suitable as well for entomovectoring in open field (11).
Another important advantage that bumblebees have over the honeybee is the fact that they perform buzz pollination (20,21). This is a very efficient pollination technique in which the flower stamens are shaken intensively by the vibrating wings of the insect, resulting in the formation of a cloud of pollen grains (23). Using this technique, bumblebees can pollinate flowers more efficiently than honeybees (22).

Case studies with honeybees Apis mellifera
The first study on entomovectoring was conducted by Peng et al. (5) for the protection of strawberry plants against B. cinerea with Apis mellifera and the microbial BCA Gliocladium roseum. This system was able to suppress B. cinerea on the flowers in the greenhouse and in the open field, except when the weather conditions were bad due to the resulting reduced foraging activity.
41_1_09_Temmermans & Smagge -pre-print 7/24 Honeybees and bumblebees were used for entomovectoring the fungus Trichoderma harzianum on strawberry plants in open fields for the control of B. cinerea (18). This entomovectoring system resulted in better control of B. cinerea than by conventional spraying. The more targeted and continuous application of the BCA at flower opening, in comparison to spraying which occurred before most flowers had opened, resulted in flowers being immediately protected at opening: the unprotected time is significantly less compared to spraying.
Furthermore, entomovectoring contributed to on average 22% more seeds with a 26 to 40% (by weight) higher yield as a result (18). Entomovectoring with T. harzianum resulted in a level of control and a yield that were at least as good or better compared to the use of commercial fungicides plus pollination (18).
In another study (7)

Transport of vectors: Impact of international import and export Current status
As important pollinators, bumblebees (Bombus spp.) have been bred and used for pollination since the late eighties. In 1987, bumblebees were first used in Belgium for the commercial pollination of tomato plants (2). Already in 2006, Velthuis and Van Doorn (2) indicated commercial pollination by bumblebees as a € 55 million industry with over a million colonies produced worldwide in 2004. Companies such as Biobest produce bumblebees for several parts of the world (Table 1).

Potential risks in importing exotic vectors
The increasing commercialisation and the associated anthropogenic movement of commercial pollinators in and outside their natural ranges can have an impact on the native bee species (31). Moreover, commercial agricultural populations can impact the local bee fauna through several mechanisms (32). For example, they are said to spread harmful pathogens to native wild bee populations. In this sense, commercial populations act as pathogen reservoirs that can contaminate wild populations. Several commercial populations of bees have already been reported to contain elevated parasite loads as reviewed by Meeus et al. (31). Furthermore, in many cases the invasive bumblebee species have an overlapping foraging activity compared to the native bumblebee species. As such, they can replace the native species through competitive exclusion (33). Both competition for floral resources and nesting sites can have negative effects on the local bee fauna (32). Finally, the interspecific mating between invasive and native bumblebee species can have a detrimental impact on the native bee species (32).
The global trade of bees facilitates the introduction of potentially invasive species into new environments (see also Goka [34], this issue). Today, regulations are often still too regional to help in solving this global problem. For example, Norway has banned introduction of commercial bumble bees and solely uses species that are local or grown in their country to protect their native species. Estonia, on the other hand, has no national risk assessment concerning pollinator introduction. The reasoning is that while use of pollinator services or entomovectoring is not common in Estonia, there has not been any need for this specific risk assessment (private communication with the Estonian University of Life Sciences).
In Brazil, the introduction of alien species is forbidden and requires a license and specific authorization from the environmental agency (private communication with the Universidade Federal da Bahia in Brazil). Bumblebees for example have been shown to escape management, resulting in an invasive species in the wild as reviewed 41_1_09_Temmermans & Smagge -pre-print 11/24 by Aizen et al. (35). Chile participates in the trade of bumblebees for commercial pollination, which is believed to have spread two alien bumblebee species, Bombus ruderatus and B. terrestris, into Argentina, which bans the import of commercial alien bumblebees (35,2). interspecific mating, the reproduction of the native bumblebee species B. ignitus and B. hypocrita sapporoensis is reduced through the production of inviable eggs (39). As a result, the replacement of the native bumblebees by B. terrestris is accelerated.
In 1880, B. terrestris was introduced in New Zealand for the pollination of red clover and merely five years later, populations were found in the wild. Subsequently the pollinator expanded its territory at a rate of approximately 90 km/year becoming the main pollinating bumblebee (32 BVT). In their legislation, the EPA encourages the development of biopesticides via shorter registration procedures and lower registration fees (40). In the EU, the approval of microbial BCAs is done at strain level by the European Commission. However, compared to chemical substances, guidance on risk assessment of microbial active substances is lacking. Also in the US, guidelines on chemical substances are more extensive than their microbial alternatives. Despite the lack of general guidance to the evaluation of microbial BCAs in the EU, the OECD Guidelines on the environmental safety evaluation of microbial BCAs are generally followed (40). Generally, the use of active microorganisms carries risk due to their potential to infect and multiply in host organisms and to produce toxic secondary metabolites upon contact with non-target organisms. These risks should be assessed, unless non-target organism exposure can be disproved (40).
Specifically for the entomovectoring system, harm to the vector is another point of attention (7). For example, Bacillus subtilis QST713, the active component of Serenade, has an LC50 of 5663 ppm for honeybees. However, honeybees directly sprayed would be exposed to approximately 8000 ppm, clearly above the LC50. Nevertheless, EPA It is clear that extra topical tests are very important to determine a system that does not harm the vector, specifically for the entomovectoring system where the vector comes in direct contact with the BCA. Furthermore, due to the increased globalisation for these emerging options, international guidelines should augment the national ones facilitating commercialization.

Conclusions
It is clear that entomovectoring combines the value of pollination with useful antipathogenic properties. Doing so, it further increases the international market for the several vector species used. Although, compared to regular pollinator transport, no extra management is needed during vector transport, misperception about the purpose, content, and risks might occur as BCAs and vectors are packed separately. It is important to provide a detailed description on the purpose for both the vector and the BCAs. Honeybees and bumblebees are the main pollinators for the entomovectoring system, and both are already treated as special cases in international transport. However, the abovementioned historic cases presenting pollinators that are also frequently used in the entomovectoring system confirm the value of developing future systems using local species, where feasible.
Despite the practical challenges in countries lacking local rearing facilities or relevant research, use of local species/populations for future entomovectoring avoids transport issues described in this thematic issue. Furthermore, these investments may help to protect pollinator biodiversity locally and also lead to higher efficiencies in the system. In the meantime, entomovectoring needs international guidelines for the registration of BCAs in order to deliver benefits sooner, including testing for (sub)lethal effects upon topical exposure.