Invasive brown marmorated stink bug (Hemiptera: Pentatomidae) facilitates feeding of European wasps and ants (Hymenoptera: Vespidae, Formicidae) on plant exudates

The brown marmorated stink bug, Halyomorpha halys, is a polyphagous species from eastern Asia, which has spread to America and Europe where it damages many crops. In recently colonized areas, facilitative interactions between H. halys and native insects are poorly investigated. In this study, we report for the fi rst time facilitation of native wasp and ant feeding by H. halys in Europe. The facilitation was related to the outfl ow of plant exudates caused by H. halys feeding on manna ash trees, where they have aggregated in response to an aggregation pheromone, which then attracted species of Hymenoptera to the infested trees. Trees other than manna ash were not involved in the facilitation between these two taxa. The species that frequently visited infested manna ash were Polistes dominula, Vespa crabro, Formica (Serviformica) cunicularia and Lasius emarginatus, while Polistes cf. nimpha, Vespula germanica, Crematogaster scutellaris and Tapinoma subboreale were occasional visitors. The numbers of wasps and ants feeding on plant exudates differed at different times in a day, with more Hymenoptera foraging in the afternoon, when more H. halys individuals were actively feeding. Facilitative interactions, such as those recorded in this study, are important for furthering our understanding of the ecology of invasive species in terms of creating sources of food for native organisms.


INTRODUCTION
When phytophagous stink bugs (Hemiptera: Pentatomidae) feed, they insert their stylets into plant tissue and suck up nutrients, which causes wounding and abortion of fruit and seeds (Panizzi, 1997;Panizzi et al., 2000). Their feeding may affect both the phloem and the xylem of a tree (Torres et al., 2010;Lucini & Panizzi, 2016).
Brown marmorated stink bug, Halyomorpha halys (Stål), is an invasive species of Pentatomidae native to eastern Asia that was fi rst detected in the United States in the 1990s and Europe in the mid-2000s and now considered one of the most damaging agricultural pests worldwide (Kriticos et al., 2017;Leskey & Nielsen, 2018). On a wide spectrum of host plants, H. halys mainly feeds on phloem (Ghosh et al., 2017), and potentially also on xylem and mesophyll/parenchyma (Serteyn et al., 2020). The damage to crops is usually due to the feeding of both immature and adult stages (Leskey & Nielsen, 2018). In some crops, H. halys infestations can result in an increase in the incidence of plant diseases (e.g. Kamminga et al., 2014;Rice et al., 2014;Paltrinieri et al., 2016;Moore et al., 2019). In the USA, H. halys is reported to feed through the bark of some species of trees, causing the outfl ow of tree c oxidase subunit I (coxI) as described by Hebert et al. (2003).
Additional observations were made in September and October 2017, and in the following two years (not included in statistical analyses).

Statistical analyses
Data on the abundance of H. halys recorded by beat sampling and visual estimates of trunk feeding activity on different species of trees were analyzed using a one-way analysis of variance (ANOVA) through the GLM procedure in SAS (ver. 9.4;SAS Institute, 2016). In this analysis, the number of H. halys recorded on different trees by beat sampling and the number observed feeding on the trunks were dependent variables, in two separate analyses in which species of tree were explanatory variables, and their effect was tested using an F test (α = 0.05). Differences among tree species were evaluated using a post hoc Tukey's test (α = 0.05). Before the analyses, data were checked for ANOVA assumptions and transformed to log (x + 1).
We assessed the relationship between the numbers of H. halys feeding on the trunks of trees and of the most common species of wasps and ants feeding on the associated exudates using linear regression. In this analysis, the mean number of H. halys feeding on trunks was the independent variable, and the number of most common species of wasps and ants the dependent variables resulting from the visual inspections of fi ve plants.
The variation in the abundance of H. halys and wasps and ants during the course of a day was assessed using the generalized linear mixed model with the MIXED procedure in SAS (ver. 9.4;SAS Institute, 2016). The different times in a day (i.e. 11:00 a.m., 2:00 p.m., and 5:00 p.m.) were the independent variable, while the dependent variables in separate analyses were the number of feeding H. halys and the presence of wasps (number of individuals) and ants (classes of abundance) feeding on plant exudates. The effect of the time of day was tested using an F test (α = 0.05) followed by post hoc Tukey's test (α = 0.05). The factor "tree" (n = 5) was included as a random effect. In this analysis, untransformed data were used.

RESULTS
In 2017, H. halys collected by beat sampling was more abundant on Fraxinus ornus L. (Scrophulariales: Oleaceae) than on the other species of trees (F 4, 320 = 619.67, p < 0.001; Table 1 (Table 1). In addition, the number of H. halys recorded feeding on the trunks of F. ornus was higher than ants) on manna ash trees by invasive H. halys. To study this phenomenon, we artifi cially induced a long-lasting infestation of H. halys in which this pest fed on trunks of trees for months, which resulted in the outfl ow of plant exudates. We investigated if the presence of wasp and ant foragers was associated with the presence of H. halys feeding on trunks of trees and if their abundance differed at different times of day.

Site surveyed and methodology
The experiment was conducted in 2017 at the experimental farm of the Department of Agronomy, Food, Natural resources, Animals and Environment -University of Padua in Legnaro,45.344124 N,11.954504 E, 8 m a.s.l.). On a group of trees of different species (Table 1) all growing close together and located within an area of about 500 m 2 , H. halys infestations were induced from June until September by using H. halys aggregation pheromone lures (Trécé Inc., Adair, OK, USA), which were installed on 5 June 2017. Lures were loaded with a synthetic analog of the aggregation pheromone, i.e. a pheromone that is known to be attractive to both sexes, including nymphs (sensu Millar, 2005) of H. halys (Weber et al., 2017). Eight pheromone lures were evenly distributed (every 5 m, maximum three lures per tree; Table 1) by hanging them in the canopies of trees three meters above the ground and renewed every three weeks in the same position. We assessed the abundance of H. halys on all the trees in the area every week (5 June -31 August 2017) by beat sampling (beating sheet, 1 m × 1 m, UM01 -Clap Net, Omnes Artes s.a.s., Bergamo, Italy) after 5.30 p.m. In addition, visual inspections of the trunk of each tree were made from 21 July to 31 August 2017 to quantify the number of H. halys feeding and the presence of other insects. Trees with pheromone lures were not included in order to avoid bias. Inspections, each lasting 15 min, were carried out at 11:00 a.m., 2:00 p.m. and 5:00 p.m. (GMT + 1) local time, every 2-4 days (13 observation days and 39 surveys in total); surveys were not carried out on rainy days. Immediately before recording the number of wasps and ants present, two minutes were spent counting the number of bark-feeding H. halys on each tree. The number of Hymenoptera observed feeding on trunks was recorded on a 1-m section of trunk 1.5 m from the ground on fi ve randomly selected trees per species. Each 1-m section was inspected, and wasps and ants actively feeding on plant exudates were counted. For ant species, the abundance was ranked in fi ve classes: 0 = absent, 1 = less than 10, 2 = 11-25, 3 = 26-40, 4 = more than 40 workers. Wasp abundance was recorded as absolute numbers. Wasps were identifi ed using Dvořák & Roberts (2006) and ants initially using a guide (Lebas et al., 2016) followed by confi rmation using mtDNA analysis, cytochrome  Fig. 1A). On F. ornus, their feeding caused the outfl ow of tree exudates from the feeding wounds, whereas no exudates were detected on other tree species. Even though some Hymenoptera were recorded on trees other than F. ornus, they were not actively feeding on trunks. The European hornet, Vespa crabro Linnaeus (Fig.  1B), the paper wasp Polistes dominula (Christ) (Fig. 1C) and Formica (Serviformica) cunicularia Latreille (Figs 1D and S1B; on all 13 days sampled) regularly visited F. ornus trunks to collect exudates. Lasius emarginatus (Olivier) workers were often recorded on F. ornus trunks feeding on exudates (on a total of eight days). In August, Polistes cf. nimpha (Christ) and Tapinoma subboreale Seifert were recorded on two and four occasions, respectively. Following this, the results for the most common wasp and ant species that fed on F. ornus (P. dominula, V. crabro, F. cunicularia and L. emarginatus) were analysed statistically.
Foragers of the German yellowjacket, Vespula germanica (Fabricius) (Fig. S1A) were recorded feeding on F. ornus exudates from September to the beginning of October 2017. While all the aforementioned species were recorded in all three years, Crematogaster scutellaris (Olivier) foragers were only recorded feeding on F. ornus exudates in 2019 (Fig. S1C).
Furthermore, P. dominula, V. crabro and V. germanica were recorded disturbing and removing H. halys in order to collect fresh exudates from the trunk (Fig. S2), but this aggressive behaviour was only recorded on a few occasions.

DISCUSSION
Here we report, for the fi rst time in Europe, facilitation of native wasp and ant feeding by invasive H. halys. These interactions are associated with the outfl ow of exudates from feeding punctures of H. halys on the trunks of F. ornus. In the USA, Fraxinus americana L. is considered to be an important host plant of stink bugs and hosts high densities of H. halys (Nielsen & Hamilton, 2009). Feeding on trunks or twigs is uncommon in stink bugs (Panizzi, 1997), but is reported for H. halys in the USA by Martinson et al. (2013) and also confi rmed in Europe.
In the area studied, the induced-massive feeding on the trunks of manna ash trees by H. halys resulted in exudate outfl ow from their feeding punctures, which attracted eight species of native wasps and ants. No wasps or ants, however, were observed feeding on the liquid faeces produced by H. halys. Despite H. halys fed on species of trees other than F. ornus, we did not observe exudate outfl ow from the feeding punctures, possibly due to specifi c features of those trees and the low stink bug numbers recorded feeding on them. In this study, high infestation levels were artificially induced by using pheromone lures, so that the results do not refl ect the true extent of the interaction between H. halys and other insects brought about by their feeding on the trunks of manna ash. Outbreaks of H. halys, however, are reported in invaded areas in Europe (e.g. Costi et al., 2017;Leskey & Nielsen, 2018;Maistrello et al., 2018). In addition, bark-feeding by H. halys is reported at other sites where attraction of V. germanica and Formica sp. on F. ornus and, with a less extent, on Acer sp. exudates following natural outbreaks of H. halys was observed in 2017 and 2019 (D. Scaccini, unpubl.). These observations indicate that facilitation of European wasp and ant feeding by H. halys may occur naturally in other areas. Similarly, the interactions between H. halys and native Hymenoptera reported in Maryland and West Virginia indicate that at least fi ve species of native wasps and ants and two cosmopolitan ants are attracted by the carbohydrate rich source exuding from the feeding wounds made by this stink bug (Martinson et al., 2013).
Nutrient requirements of wasps and ants include carbohydrates, usually obtained from (extra)fl oral nectaries, honeydew, fruit, plant sap and other products, as well as anthropogenic compounds. Sugars are used as an energy source by wasps (Raveret Richter, 2000) and in metabolism and sperm production by ants (Wäckers et al., 2005;Blüthgen & Feldhaar, 2010).
Phloem sap is a food rich in sugars (Douglas, 2006) and, as reported for Oleaceae in general (Wäckers et al., 2005), Fraxinus exudates contain mannitol, fructose, glucose and other monosaccharides, together with oligosaccharides such as mannotriose and stachyose (Caligiani et al., 2013). Vespa crabro is known to feed on tree sap (Yoshimoto & Nishida, 2009) and Archer (2014) states that V. crabro actively wounds twigs of ash and lilac trees in order to cause an outfl ow of sap. Ants foraging activity on manna ash tree exudates is not surprising because sugar is a common part of the diet of Formicinae such as F. cunicularia (Akyürek  , 2016;Novgorodova & Ryabinin, 2018) and Lasius species (Madsen et al., 2017), in which the workers have receptors that possibly perceive carbohydrates (Tinti & Nofre, 2001). Crematogaster scutellaris is omnivorous and may attack and eat H. halys (Castracani et al., 2017), but in this study, it was only recorded foraging for plant exudates.
Halyomorpha halys feeding activity varied during the course of a day being higher in the afternoon than late in the morning. Indeed, H. halys activity increases with increase in temperature, but probing activity stops above 26.5-29. 6°C (Wiman et al., 2014), development ceases at 35°C and it dies at higher temperatures Aigner & Kuhar, 2016;Scaccini et al., 2019).
More wasps and ants were recorded feeding on F. ornus exudates in the afternoon than in the morning. Indeed, when it is warm, the rate of delivery of sugars by wasp foragers to their nest is higher than at lower temperatures, as reported for V. germanica (Jandt et al., 2010). Similarly, ant foraging activity is affected by environmental factors such as temperature, light intensity and other physical and biotic factors (Traniello, 1989), and at least in boreal forests, it generally increases in warm periods (Domisch et al., 2009).
Furthermore, on manna ash trees Vespidae disturbed H. halys feeding on the trunks. In fact, the aggressive behaviour of wasps toward other insects feeding on plant exudates was quoted by Wilson (1926) and, more recently, by Yoshimoto & Nishida (2009) and it dominates the hierarchies of insects feeding on plant exudates. Further investigations on aggressive behaviour, however, are needed for a better understanding of the plant exudate-feeding insect community.
The outfl ow of plant exudates that result from the feeding of phytophagous insects is known to attract different species of Hymenoptera in other regions (Yamazaki, 2007;Martinson et al., 2013), hence considered herbivores under an ecological point of view (Jones, 1994;Raveret Richter, 2000;Yoshimoto & Nishida, 2009;Iakovlev et al., 2017). Non native species can increase the availability of this sugary food source and create a novel feeding niche for native organisms (Rodriguez, 2006) that feed on tree exudates induced by phytophagous species. The positive interactions reported here are important for further understanding the ecology of invasive non native organisms, which induce plants to liberate exudates that are as attractive for native Hymenoptera as other sources of sugar from plants.