**3. Impacts**

In Europe, *V. v. nigrithorax* is considered invasive, both for its expansion capabilities at European scale [44–46] and the impacts that it could produce by preying on honey bees and native

insects [35,47–49]. The species can cause serious damage and imbalances to biodiversity and ecosystems in areas where it has been introduced. This is aided by *V. v. nigrithorax*'s high reproductive rate, high dispersal ability, broad diet, wide habitat preference, superior competitive ability, and most importantly, multiple mating of its queens [13,50,51].

Although the species is not considered in Europe more dangerous than native hornets and wasps [52], it can cause problems to human-health and several accidents and some fatal events were recorded [53]. Moreover, by frequently establishing colonial nests in urban areas, *V. v. nigrithorax* could generate social impacts due to citizens' perception of fear of possible stings [54–56]. *V. v. nigrithorax* causes multiple threats, however the extent of the impacts produced has not ye<sup>t</sup> been documented and quantified exhaustively. Despite this lack, it is possible to assess the components and/or the activities that are affected or may be impacted by this hornet. From the analysis of the research conducted so far in Europe it is possible to reckon three major negative impacts that the species may create:


#### *3.1. Damage to Beekeeping*

The hornets hunt foraging honey bees returning to their colonies by hovering in front of the hive entrance, grabbing the honey bees in flight, and killing them with their jaws. The thorax is then selected, for the high protein content provided by the flight muscles, and transported to the nest to be fed to the larvae.

The intense predatory activity of *V. v. nigrithorax* towards honey bees can generate a decrease in the strength of the honey bees colonies and the subsequent collapse of the family. This is due to the increase in the number of foraging bees which do not come back in their hive with a consequent increase of the overall probability of homing failure and finally with the disruption of colony foraging activities, which leads to complete foraging paralysis [57]. This primarily generates economic damage to the beekeeping sector, as well as a decrease in the number of honey bees in the environment, resulting in a decline in the ecosystem pollination service.

In some European regions, predation of *V. v. nigrithorax* has resulted in the loss of almost 50% of bee hives. In the south-west of France, beekeepers reported losses of between 30% and 80% of honey bee families, resulting in poor production of honey and other beehive products. In 2010 in Gironde (France), due to *V. v. nigrithorax*, the Union Nationale pour l'Apiculture Francaise declared that 30% of bee hives were destroyed or weakened [58]. In Western Liguria (Italy) the authors of the present review (unpublished results) have found an increase of 18% in winter colony losses in areas where *V. v. nigrithorax* is not controlled.

The costs incurred both for the implementation of public information campaigns and for the destruction of *V. v. nigrithorax* nests are relevant economic issues. In 2011 in France, the beekeepers' organization Groupement de Dèfense Sanitaire des Abeilles (GDSA) coordinated the destruction of more than 1,000 nests in Aquitania, while a private company destroyed about 500 nests in the Toulouse area. The total cost of these interventions can be quantified to more than 165,000 euros.

Leza et al. [21] demonstrated that the presence of *V. v. nigrithorax* produces an increase of oxidative stress in honey bee workers under field conditions. This leads to a higher expression and activity of antioxidant enzymes and mitochondrial-related genes and higher lipid oxidative damage in the individuals of the colony exposed to this predator. Other authors reported that other stressors, like herbicides or migratory management, could increase lipid peroxidation in honey bees [59] suggesting that these situations, along with the presence of *V. v. nigrithorax*, could affect honey bees' health [21].

The apiaries are a very attractive source of food for *V. v. nigrithorax*, because there is a high concentration of honey bees. Studies performed in France have demonstrated that in urbanized environments, where the concentration of apiaries is high, the diet of *V. v. nigrithorax* is composed for almost 70% of honey bees and other similar species (Apoidea) [60]. Monceau et al. [49] monitored the predation of *V. v. nigrithorax* on apiaries. In an apiary with six beehives, in the sampling period they caught a total of 360 workers, and most of these visited the apiary daily. This indicates that once the species has identified an important protein source such as an apiary, it visits the site every day, probably because of a greater success of predation. Of the six beehives monitored in the season, one was completely destroyed, while in the other five the size of the colonies halved. In addition, five *V. v. nigrithorax* nests were discovered within 1 km from the experimental apiary; so it is likely that apiaries were attacked by individuals from different colonies.

The colonies of *Apis mellifera* manifest a certain defensive ability towards *Vespa crabro*, the European native hornet, but fail to implement effective defensive behaviours towards *V. v. nigrithorax*, having had no opportunity to co-evolve with this predator. In the Asian regions, where the local bee species, *Apis cerana*, has co-evolved with *V. v. nigrithorax*, the honey bees have instead developed very effective defense techniques, resulting in suffocation and heating of the predator (balling), and the formation of a compact agglomeration of honey bees on the flying board [61]. *A. mellifera* is also able to curl up hornets that rest on the flying board of hives, but this behavior does not reach large percentages of success.

At the end of summer, when the colonies of *V. v. nigrithorax* are very populous, hornets can ge<sup>t</sup> to besiege the hives and penetrate inside them annihilating the colonies of honey bees. The use of doors with passages less than 5.5 mm in diameter can prevent the entrance of hornets and delay the definitive collapse of the colonies, but if the beekeeper does not intervene to eliminate hornets, the honey bees cannot ge<sup>t</sup> out and the colony is destined to collapse (Figure 3).

**Figure 3.** *V. v. nigrithorax* in hunting activity in front of a hive (left) and heavy attack of hornets on the flying board of a hive (right).

As it often happens in many species of insects, climatic conditions, especially temperature and humidity, affect the predator's activities. In the case of *V. v. nigrithorax*, the increase in the efficiency of predation, which is most evident in the middle hours of the day, would be the result of an increase in temperatures and the level of solar radiation [38].

#### *3.2. Impact on Ecosystem*

The predatory activity of *V. v. nigrithorax* has a negative impact on insect communities, reducing their abundance and may cause damage to local biodiversity even at the ecosystem level. The predation pressure known in apiaries since the month of July may have similar effects also on other pollinating insects, creating a decline in pollination effectiveness. In fact, besides honey bees, *V. v. nigrithorax* preys

on other Hymenoptera, including different species of wild bees and other Vespidae (wasps in general), but also Diptera (flies and mosquitoes), butterflies species and other insects. Species preyed upon by *V. v. nigrithorax* and their proportion varies according to the prey availability in the environment. A French study showed that in an urban environment, *V. v. nigrithorax* preys mostly honey bees and other Apoidea (66% of the diet), while in a woodland environment, bees and other Apoidea drop to 33% and Diptera increase to 32% [60] (Figure 4).

**Figure 4.** *V. v. nigrithorax* prey spectrum: Preliminary results in three different environments [60].

#### **4. Monitoring and Surveillance Systems**

Monitoring the spread of an invasive alien species is crucial to plan appropriate managemen<sup>t</sup> actions and activities to limit its expansion. Only monitoring and surveillance strategies permit to assess the presence of the species on the territory and identify the areas of expansion or new invasive outbreaks. Since *V. v. nigrithorax* is particularly attracted by honey bees, it is important to involve beekeepers and beekeeper associations to maximize the efficacy of monitoring strategies, together with the contribution of all interested citizens.

An early detection of *V. v. nigrithorax* in areas far away from the expansion front allows to perform a rapid response aimed to remove these isolated populations before the settlement of the species [62]. In fact, *V. v. nigrithorax* queens might be accidentally transported by human activities in very remote areas, where these insects can give rise to new colonies and populations [26]. An early warning and rapid response system (EWRRS) for *V. v. nigrithorax* is based on three key moments (Figure 5).

Results of EWRRS are the rapid detection of the species and the readiness of intervention, which increases the probability of destroying the colonies before the birth and the mating of the future founder queens. This increases the probability of success in the containment of *V. v. nigrithorax*. Key aspects of an effective managemen<sup>t</sup> strategy are: Simplicity of the procedures; rapid intervention; exportability on a national and international scale; economic sustainability.

Different monitoring methods for *V. v. nigrithorax* exist: Direct observations of hornets in apiaries or on flowers and the use of traps.

Several trap models have been proposed to catch *V. v. nigrithorax* adults; basically, they can be reduced to bottle, funnel, and sticky traps or to a combination of them.

In addition to proprietary baits, many types of self-produced carbohydrate or protein baits can be used. Sugar based baits include beer, vinegar, grenadine, acetic acid, fermented honeycomb juice, honey, different type of sweeteners mixtures, etc. [40]. They are better used between February and May, so to catch the founder queens when they begin the construction of the primary nests, and from August until November, to detect the presence of the species in new areas or to catch the reproductive adults. Demichelis et al. [24] recommend the use of lager beer (0.33 litres with 4.7% alcohol), because it is attractive for the hornets, inexpensive, and selective towards honey bees (Figure 6).

**Figure 5.** Early warning and rapid response system for *V. v. nigrithorax*.

**Figure 6.** Bottle trap with lager beer [24].

During colony development, between June and August, protein baits (meat, fish, etc.) can be used. In addition, Rodríguez-Flores et al. [18] highlighted that elevation and meteorological factors influence the effectiveness of bait trapping.
