BioInvasions Records (2019) Volume 8, Issue 3: 521–529

CORRECTED PROOF

Rapid Communication The Balkans invaded – first record of communa LeSage, 1986 (Coleoptera: Chrysomelidae) in Croatia

Mladen Zadravec*, Barbara Horvatić and Paula Prpić Association Hyla, Lipovac I no. 7, HR–10000 Zagreb, Croatia *Corresponding author E-mail: [email protected]

Citation: Zadravec M, Horvatić B, Prpić P (2019) The Balkans invaded – first record Abstract of LeSage, 1986 (Coleoptera: Chrysomelidae) in Croatia. Ophraella communa was found for the first time in Croatia. After the accidental BioInvasions Records 8(3): 521–529, discovery, further work was done to ascertain its extent of occurrence, density of https://doi.org/10.3391/bir.2019.8.3.07 adults and severity of host plant attack. Additional data pertaining to its current Received: 4 January 2019 status in Croatia were also gathered. It was found on ten locations, with the highest Accepted: 26 April 2019 recorded density in the SE part of Lonjsko Polje Nature Park. The damage levels of Published: 10 July 2019 attack were never as high as in Italy or Switzerland. It is unknown whether this species was introduced intentionally or accidentally. Future work should focus on Handling editor: Angeliki Martinou monitoring the spread and influence of this alien species in Croatia. Thematic editor: Stelios Katsanevakis Copyright: © Zadravec et al. Key words: introduced alien species, distribution, extent of occurrence, biological This is an open access article distributed under terms control, of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0).

OPEN ACCESS. Introduction The introduction of alien species has for some time been regarded as an ecological and economical problem that is getting worse as time goes on. It is even regarded by some as being a significant component of ecosystem changes worldwide (Bosio et al. 2014; Cote et al. 2010; Katsanis et al. 2013; Müller-Schärer et al. 2014; Raak-van den Berg et al. 2012; Vitousek et al. 1996). Therefore, it is of utmost importance to closely monitor the spread and influence of the invaders on native species, in order to devise mitigation and restorative actions. Ophraella communa LeSage, 1986 is a North American leaf which mainly feeds on , Ambrosia artemisiifolia L. (SMARTER 2014). Outside its native range, it had first been detected in Japan, then in China, in the late 1990s and early 2000s, respectively (Takizawa et al. 1999; Zhou et al. 2010a). The first European records originate from 2013 in northern Italy and southern Switzerland (Müller-Schärer et al. 2014). In 2017 it was found in Slovenia, where it probably expanded naturally from Italy one or two years before (Seljak 2017). Currently it is present only in the westernmost part of the country (Seljak 2017).

Zadravec et al. (2019), BioInvasions Records 8(3): 521–529, https://doi.org/10.3391/bir.2019.8.3.07 521 Ophraella communa in Croatia

Figure 1. Map of investigated locations, showing the presence/absence of Opraella communa LeSage, 1986 in Croatia. Inserts to the right – stages of O. communa: A – adult, B – pupa, C – L3 larva, D – eggs. For details see Supplementary material Table S1. Photos by Mladen Zadravec.

Due to its voracious appetite for Ambrosia artemisiifolia (), whose pollen is highly allergenic, it became the focus of several studies, both in the lab and in the field, to ascertain its potential as a safe biocontrol agent (Teshler et al. 2004; Zhou et al. 2010a, b; Zhu et al. 2012). Due to fears of it switching host plants, especially in regard to sunflowers, investigations into that matter were performed (Bosio et al. 2014; Zhou et al. 2011). Here we present the first records of Ophraella communa in Croatia, indicating this species has expanded to the Balkan peninsula. Its current distribution has been mapped and some additional notes regarding its current status in Croatia are also given. Finally, the development of a monitoring methodology is briefly suggested.

Materials and methods The field work was carried out during the summer of 2018 on 41 locations harbouring ragweed (Supplementary material Table S1, Figure 1). Locations were chosen either because they were already known to have A. artemisiifolia based on earlier unrelated observations, or by examining satellite imagery for those which could potentially host the plant. In the latter case, fields and field edges, roadsides, canal edges and similar ruderal/marginal

Zadravec et al. (2019), BioInvasions Records 8(3): 521–529, https://doi.org/10.3391/bir.2019.8.3.07 522 Ophraella communa in Croatia

habitats were chosen, as they were the most likely locations to harbour A. artemisiifolia – and all chosen locations did. The distance between locations was always at least 500 m. Each location was visited at least once and between 15 and 60 minutes were spent working per location. On 33 of them, at least one patch of A. artemisiifolia per location was first inspected visually for the presence of the . If that was negative, sweep netting was performed with a sweep net (diameter = 35 cm) on patches with a prevailing plant height of over 20 cm. On some locations the number of sweep net strokes was pooled for all patches there, due to the number of strokes not being recorded separately for each of them. The prevailing plant height was measured in 20 cm intervals. Patch sizes and shapes varied, depending on the terrain configuration and the way ragweed sprouted there. The minimum distance between each patch was five metres, while the border area between them always had to be completely void of ragweed plants. The incidence of attack was estimated according to Müller-Schärer et al. (2014). The neighbouring plants, especially Asteraceae, were also checked for damage. When observed, each egg cluster was assigned a sequential number, its position on the plant was recorded and they were photographed, so that the number of eggs may be counted later, to provide additional data. The remaining eight locations were also checked for the presence of O. communa, but the adherence to the described methodology was somewhat lax (Table S1). To ascertain the density of adults, a part of each tested patch which did contain adults was randomly selected for sweep netting, preferably with at least ten strokes. The sweep net was emptied and all caught adults of O. communa were counted one by one, after which they would be released on the same patch. This was done only in July and August, after the methodology was defined. The density per patch was calculated by dividing the number of sweep netted adults by the number of sweep net strokes. These were then averaged to get the density per location. The point of origin is proposed based on the geographic position of positive locations and the average adult densities. The proposition was tested in MS Excel 2016, by calculating the Pearson’s correlation coefficient. The were identified using a combination of SMARTER (2014) and Warchałowski (2010). The maps were created in ArcMap 10.2.2 (ESRI). All collected specimens are stored in the private beetle collection of the first author (Zagreb). Coordinates for all locations were recorded using a Garmin eTrex 30x GPS device. All photographs were taken using a Canon PowerShot SX710 HS digital camera. The coordinates are given in the WGS84 coordinate system, as decimal degrees.

Results Distribution in Croatia and degree of damage Ophraella communa was found on a total of ten locations. The results of the study, including the prevailing plant height, total number of sweep net

Zadravec et al. (2019), BioInvasions Records 8(3): 521–529, https://doi.org/10.3391/bir.2019.8.3.07 523 Ophraella communa in Croatia

strokes, prevailing plant heights, degree of damage on plants, and recorded beetle stages at each site, are given in Figure 1 and Table S1. It was first discovered by accident on location 9, after three passes with a sweep net were performed on a small semi-shaded patch of herbaceous vegetation by a macadam road, containing several ragweed plants. Several dozen individuals were discovered in the sweep net. Two larger nearby patches of A. artemisiifolia were then checked, with adults and eggs being found on all of them, both by visual observation and by sweep netting. When revisited in July, the damage to ragweed plants was greater. On locations 11, 12, 15 and 16 various stages were found by visually inspecting the patches, in less than two minutes per location, hence sweep netting was not performed. Of the four investigated patches on location 13 in June, only one was partly infested on the first date, with pupae and larvae. There were no findings on the others, even after sweep netting. This location was revisited two days later, when even more pupae and larvae were observed. Adults and eggs were also seen. Additionally, two adults and several clumps of eggs were found on a second patch, 8 m away from the first one. The third and fourth patches, approximately 60 and 100 m away from the first one, respectively, still yielded no findings. By July the four previous and two new patches at this location were all infested by one or more stages of Ophraella, despite being mowed between the visits. Only two adults were found on location 14, after 20 minutes of visually inspecting the plants in June. It is possible the beetles were simply dormant and in hiding, since that day was overcast, and the location was inspected just before the onset of heavy rainfall. When this location was revisited in July, it was discovered it had been recently mown, but ragweed plants already re-sprouted. Several of them had feeding marks on the leaves, but no beetles were found, even after 150 passes with a sweep net on two patches. Four days later, almost all stages were found, and even more plants were damaged on both patches (but see discussion for adult densities). Location 17 is designated as one where the ragweed leaf beetle “may be present” since the damage to the plants resembled that of Ophraella and that the beetles may have been missed similarly to what occurred on location 13.

Host plant specificity, number/position of eggs, density of adults The beetle stages were observed all over A. artemisiifolia, but not always at the same time per visit to a location (see Table S1). Adults, pupae and larvae were on both the upper and underside of the leaves, on the stems and even in buds. One pupa was found on a leaf of Plantago sp. growing adjacent to a ragweed, found on location 13, and one adult found on an unidentified Asteraceae growing amongst ragweed plants on location 16. The Plantago leaf was undamaged, therefore it is assumed the larva fell off the ragweed plant and simply pupated there. The unidentified Asteraceae

Zadravec et al. (2019), BioInvasions Records 8(3): 521–529, https://doi.org/10.3391/bir.2019.8.3.07 524 Ophraella communa in Croatia

Table 1. Densities of adults per patch and average densities per location. Loc. no. Patch no. Date No. of strokes No. of beetles Density Average Density 7 1 25.08.2018 20 4 0.2 0.15 2 25.08.2018 10 1 0.1 8 1 25.08.2018 10 29 2.9 3.25 2 25.08.2018 10 36 3.6 9 2 18.07.2018 10 36 3.6 3.3 2 18.07.2018 3 9 3 10 1 18.07.2018 10 165 16.5 9.45 2 18.07.2018 10 24 2.4 13 4 18.07.2018 10 4 0.4 0.25 6 18.07.2018 10 1 0.1 14 1 18.07.2018 20 0 0 0.1 2 18.07.2018 10 2 0.2

showed no signs of damage by the beetles, despite the adjacent ragweed plants showing moderate damage. Eggs were counted in a total of 124 egg clusters on locations 7 (27 clusters), 8 (30), 9 (51), 13 (7), 14 (7), 15 (1), and 16 (1). The eggs were deposited in the apical, central and basal zones of leaves of A. artemisiifolia, both on the upper- and underside, usually in a single cluster, but also individually or in two to five smaller adjacent clumps, with 1–40 eggs (average: 12) per cluster. Adult beetle densities were investigated on 18 July and 25 August. However, this could not be done on all locations/patches due to very recent mowing or insufficient sizes of the patches to accommodate sweep netting at those times. The densities per patch and average densities per location are given in Table 1.

Point of origin and possible methods of introduction The geographical position of investigated locations, decreasing level of attack of A. artemisiifolia by O. communa, and adult densities towards the northwest, north, and southeast within Lonjsko Polje Nature Park, and to the northeast towards Nova Gradiška, indicate that the point of introduction of O. communa may be at location 10, or somewhere in its immediate vicinity (Figure 2). The Pearson’s correlation coefficient for average density and distance from assumed point of introduction is −0.8542, which supports this hypothesis (p = 0.004).

Discussion Although it was reasonable to expect the arrival of O. communa in Croatia, its point of discovery is surprising, since it would be expected to arrive from the west, after it expanded eastwards from Italy through Slovenia (Müller-Schärer et al. 2014). However, due to its current distribution in Slovenia, this seems unlikely (Seljak 2017). The incidence of attack can vary between sites (Müller-Schärer et al. 2014), which could lead to false conclusions if the number of investigated sites is low or they are visited at a wrong time (see results for location 14). Since the assumed point of

Zadravec et al. (2019), BioInvasions Records 8(3): 521–529, https://doi.org/10.3391/bir.2019.8.3.07 525 Ophraella communa in Croatia

Figure 2. Average densities of adults per location and estimated current area of occurrence of Ophraella communa in Croatia. Red numbers – average densities per location, black numbers – location numbers (see Supplementary material Table S1).

introduction is not near any major pathways, e.g. motorways or railways, nor does it appear to be a location with a lot of frequent traffic, it is not immediately apparent how it may have arrived. But location 10 does have a hunters’ blind and hunting is popular in the area – it is possible then that Ophraella was brought here, accidentally or deliberately, by foreign hunters who came from Italy/Switzerland/Slovenia where Ophraella is present, or by local hunters who returned from a hunting trip from those countries, where they spent time in locations where Ophraella is present. Several individuals could have easily and unknowingly ended up in the hunters’ vehicles and among their equipment, like bags and cases. Small can be difficult to notice; therefore such a means of dispersal is not that far-fetched (Eritja et al. 2017). A similar situation was observed by the second author, who found another leaf beetle – Chrysolina americana Linnaeus, 1758, in Oroslavje, north-west of Zagreb, immediately after her parents returned from a holiday in the Mediterranean part of Croatia. This Mediterranean species was found in a bag, among clothes taken on the holiday (B. Horvatić, pers. obs., 2017). Also, the first author discovered one live specimen of Ophraella inside his car, several hours after visiting several of the

Zadravec et al. (2019), BioInvasions Records 8(3): 521–529, https://doi.org/10.3391/bir.2019.8.3.07 526 Ophraella communa in Croatia

investigated locations in Lonjsko Polje where the presence of the ragweed beetle was confirmed, even though extra care was taken not to accidentally transfer any on equipment or clothing (M. Zadravec, pers. obs., 2018). Ophraella communa was found quickly, within at most a couple of minutes of visually inspecting , on most locations – but not always. Locations where the beetle is assumed to be present, e.g. because of observed damage on the plants, should be revisited at least once or twice more, to try and confirm this. The cause and speed of the apparent disappearance and subsequent reappearance of the beetles like on location 14 should be investigated further. The observed damage to plants on all sites in June was never as severe as to prevent all the plants in a particular patch from flowering, like in Italy, Switzerland and China (Müller-Schärer et al. 2014; Zhou et al. 2014), except maybe the first patch on location 10. An increase in damaged plants was observed in July on several locations, but it was still medium at most, and neither rapid nor markedly increased as in Italy and Switzerland (Müller-Schärer et al. 2014). This could indicate that this is just the beginning of the invasion, similar to Slovenia (Seljak 2017). If that is the case, this presents an opportunity to monitor the spread and influence of the invasion by O. communa within Croatia, as was done with Diabrotica virgifera virgifera LeConte, 1858 (Igrc Barčić and Maceljski 1997; Igrc Barčić et al. 2003; Lemic et al. 2013; Benítez et al. 2014). Another invasive alien species widespread in Croatia (Mičetić Stanković et al. 2010), Harmonia axyridis Pallas, 1773 (Coccinellidae), has been shown to feed on the ragweed leaf beetle (Moriya et al. 2002). This could play a part in explaining why the ragweed leaf beetle population is not as large as in Italy or Switzerland, but it cannot be the sole reason. Especially since this year the population size of H. axyridis in Lonjsko Polje, despite never being calculated, appears to be substantially lower than in previous years (M. Zadravec, personal obs., 2016–2018). However, three native coccinellid species: one rarely predatory – Tytthaspis sedecimpunctata (Linnaeus, 1758), and two always predatory – Hippodamia variegata Goeze, 1777 and Propylea quatuordecimpunctata (Linnaeus, 1758), were found on the same plants as the ragweed leaf beetle – they could be potential predators of eggs and early larval instars. As the study was conducted over just a few days over a span of approximately two months, the additional data gathered, e.g. egg placement, cannot serve as a basis for estimating phenology patterns. However, they can still be of value, since it represents the first ever data gathered about the ragweed beetle in Croatia. The gathered data show that there are definitely more than two generations per year in Croatia. Three to four generations are expected in Italy (Müller-Schärer et al. 2014), therefore it could be the same here also, due to favourable climate conditions in large parts of the country (Zaninović 2008).

Zadravec et al. (2019), BioInvasions Records 8(3): 521–529, https://doi.org/10.3391/bir.2019.8.3.07 527 Ophraella communa in Croatia

Due to the proximity of the positive sites to the border with Bosnia and Herzegovina, and ragweed being widespread there (Kovačević et al. 2015), it could soon be expected in that country also, if it has not already become established. The plant is widespread in Croatia’s northern and eastern neighbours (Storkey et al. 2014), so they too should be on the lookout for the ragweed beetle. A monitoring programme based on the methodology in this study should be developed and tested, in order to better track the spread and influence of O. communa.

Conclusions The arrival of O. communa in Croatia, since its discovery in Italy and Switzerland, was expected. Targeted surveys should be carried out both to the north, west and east of Lonjsko Polje, to determine if it is more widespread, or if this is just the beginning of the invasion. The methodology used in this research can provide a basis for a monitoring protocol, which could then be performed on fixed monitoring stations distributed all over the country, to monitor the beetle’s expansion and influence. This is especially true for the areas around sunflower fields. It would be interesting to investigate whether the mostly low densities of adults are the result of predation, disease, or a combination of both.

Acknowledgements

Our thanks go to Domagoj Kanjković, Daria Kranželić, Đurđica Majetić, Mario Zadravec and Vesna Zadravec for their assistance during the field work, and to Dina Hlavati for sharing her data for the location in Našice. Funding for the Lucanus cervus monitoring project [„Monitoring jelenka (Lucanus cervus) u PP Lonjsko polje u 2018. godini“, project leader: Mladen Zadravec, Class: 641-02/18-01/06, Registry/Docket number: 2176-144-05/02-18-1] was provided by the Public Institution Lonjsko Polje Nature Park. We also thank the reviewers for their helpful comments and suggestions.

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Supplementary material The following supplementary material is available for this article: Table S1. Investigated locations and patches, with the number of sweep net strokes performed, prevailing plant heights, degree of damage and recorded beetle stages per location/patch in Croatia. This material is available as part of online article from: http://www.reabic.net/journals/bir/2019/Supplements/BIR_2019_Zadravec_etal_Table_S1.xlsx

Zadravec et al. (2019), BioInvasions Records 8(3): 521–529, https://doi.org/10.3391/bir.2019.8.3.07 529