New records and updates on alien Auchenorrhyncha species in Serbia

SUMMARY During a 2019 survey of the Auchenorrhyncha fauna in Serbia, three alien species were recorded for the ﬁ rst time: a leafhopper species of Asian origin Orientus ishidae (Cicadellidae, Deltocephalinae) and two North American species, Acanalonia conica (Acanaloniidae) and Erasmoneura vulnerata (Cicadellidae, Typhlocybinae). Besides these ﬁ rst ﬁ ndings, the presence of the leafhopper Phlogotettix cyclops (Cicadellidae, Deltocephalinae) was conﬁ rmed in Serbia after its initial record more than a century ago. Other allochthonous species already known to be present in local entomofauna were also found: Japananus hyalinus (Cicadellidae, Deltocephalinae), Scaphoideus titanus (Cicadellidae, Typhlocybinae), Stictocephala bisonia (Membracidae) and Metcalfa pruinosa (Flatidae). The newly detected Auchenorrhyncha species in Serbia, O. ishidae, A. conica and E. vulnerata, as well as Ph. cyclops, are recognized as either tentative or true grapevine pests in Europe that cause physical damage or induce phytoplasma-borne diseases. Our ﬁ ndings reveal a potential phytosanitary risk that can emerge in Serbian vineyards in upcoming years and support refocused research of Auchenorrhyncha diversity in natural habitats, rather than in agroecosystems and their immediate surroundings.

Over the past few years, a new leafh opper linked to FD phytoplasma has been detected in Europe, the mosaic leafh opper Orientus ishidae (Matsumura, 1902) (Cicadellidae, Deltocephalinae) (EPPO, 2015). Aft er it was fi rst detected in Italy, Switzerland and Germany in the early 2000s, this species of Asian origin quickly spread across the continent by moving north towards the Netherlands and spreading eastwards to Romania (Günthart & Mühlethaler, 2002;Nickel & Remane, 2003;Guglielmino, 2005;Den Bieman & Van Klink, 2015;Chireceanu et al., 2017). Interest in O. ishidae grew aft er FD phytoplasma was detected in European populations in vineyards and its vectoring potential was experimentally confi rmed (Mehle et al., 2010;Lessio et al., 2016). Furthermore, this species was already a suspected vector of 'Candidatus Phytoplasma pruni' (16SrIII) in the USA (Rosenberger & Jones, 1978). Th e presence of Nearctic species Acanalonia conica (Say, 1830) (Acanaloniidae) in Europe was fi rst reported in Italy, and soon aft er that detected in Switzerland, Slovenia and Romania (D'Urso & Uliana, 2006;Trivellone et al., 2015;Chireceanu et al., 2017;Seljak, 2018). Although widely considered as a polyphagous species and not related to crops, A. conica was observed ovipositing on grapevines in the USA (Wilson & McPherson, 1981). In Europe, it was recorded in urban areas, natural habitats and associated with several arboreal, shrubby and herbaceous plants bordering vineyards, but not on grapevines (Nicoli Aldini et al., 2008). Th e presence of another Vitis-related leafh opper, Erasmoneura vulnerata (Fitch, 1851) (Cicadellidae, Typhlocybinae) was also observed in Italy for the fi rst time, followed by records from Slovenia (Duso et al., 2005;Seljak, 2011). Th is typhlocybin species from the temperate North America (Dietrich & Dmitriev, 2006) completes two or three generations per annum in Europe and successfully colonizes several European grapevine cultivars, showing a pest potential (Duso et al., 2019).
Th is study focused especially on verifying the presence of the leafh opper Phlogotettix cyclops (Mulsant & Rey, 1855) (Cicadellidae, Deltocephalinae), which has been trending recently in phytoplasma epidemiological research as an inhabitant of European vineyards and a potential FD vector (Chuche et al., 2010;Chireceanu et al., 2017;Strauss & Reisenzein, 2018;Plavec et al., 2019). Despite rising concerns over this leafh opper as an invasive Asian pest that spreads across Europe, its presence in several European countries has been documented for more than a 100 years (Metcalf, 1967). Although occasionally perceived as an allochthonous species spreading in European fauna (Chuche et al., 2010), it is important to highlight that some authors identify Ph. cyclops as a palearctic/amphiboreal species and only representative of the genus Phlogotettix Ribault, 1942 in Europe (Gnezdilov, 2003;Meshram et al., 2015). Th e fi rst traceable record of Ph. cyclops in Serbia was made by Horváth (1903) and it specifi ed Niš as the collecting locality, but without plant or habitat data.

MATERIAL AND METHODS
Insects were collected over a seven-month period (from March to September) in 2019. Th ree types of habitats were surveyed: (1) urban area of Novi Sad in order to detect species associated with ornamental plants and vegetation adapted to urban habitats; (2) vegetation near the Belgrade Customs Offi ce Terminal, and areas along the A1 motorway in Serbia (part of the European route E-75) as a front row barrier for species that can be spread by means of transport, and (3) undisturbed habitats of the Special Nature Reserve Zasavica (SNR Zasavica), SNR Deliblatska peščara, and Stara planina Nature Park (NP Stara planina), rich in diverse native vegetation. Sampling was performed by sweep nets and mouth aspirators for precise collecting from specifi c plant species and supplemented with insect light traps to enhance the sampling rate of species that are low in abundance, arboreal or hard to reach. Swept specimens were immediately placed into 2 ml plastic tubes (Sarstedt, Germany) fi lled with 96% ethanol, while specimens attracted by light traps were transferred the following morning. Th e insect material was then transported in a portable cooler (10°C) and stored in a laboratory refrigerator at 4°C until further analysis. Collected samples were examined under a stereomicroscope (Leica MZ7.5) and assigned to specifi c species based on male genitalia. Photographs of the specimens were made using an iPhone 6S phone camera attached to a stereomicroscope, while photographs of specimens in the fi eld were taken with a Canon SX160 IS Camera. Th e collected and examined insect material was stored with corresponding aedeagi in glycerol and deposited in Marko Šćiban's private entomological collection.

RESULTS
Th e collected specimens were sorted based on the specifi c plants they were found on and/or collecting sites. Th ey were assigned to specifi c species based on their outer morphological features and male genitalia. Of more than 200 Auchenorrhyncha species identifi ed during this survey (data not shown), three alien species, previously unknown in the Serbian fauna, were discovered: O. ishidae, A. conica and E. vulnerata (Figures 1 and 2). Light traps set in Novi Sad and SNR   (Guglielmino, 2005).
Remarks: Identity confi rmed based on male genitalia according to Guglielmino, (2005 Remarks: A. conica is the only member of the fam. Acanaloniidae present in Europe with distinctive bright green coloring and conical vertex; male genitals were examined according Freund & Wilson (1995).
Distribution: USA, Canada, Mexico, Italy, Slovenia. Remarks: Identity was confi rmed based on male genitals according to Seljak (2011); all specimens had typical red R1 vein characteristic for summer populations, but the collecting date indicated that they could belong to the second generation (Duso et al., 2019).

DISCUSSION
Th e current faunistic survey has revealed the presence of three alien Auchenorrhyncha species, so far unknown in Serbian entomofauna: O. ishidae, A. conica and E. vulnerata, and confi rmed the presence of Ph. cyclops in Serbia. Frequent fi ndings of J. hyalinus, S. titanus, S. bisonia and M. pruinosa in SNR Zasavica and numerous other sites (data not shown) have indicated successful naturalization of these invasive species in Serbia. Th e newly detected species were attracted by light traps, already proven to be helpful in faunistic studies in urban and suburban areas (D'Urso & Uliana, 2006;Koczor et al., 2013). Arrival routes of the recorded allochthonous species are currently unclear, especially knowing that alien Auchenorrhyncha benefi t from agricultural and ornamental plant trade and can migrate signifi cant distances aft er their initial intrusion (Mifsud et al., 2010). Th e occurrence site of A. conica in Novi Sad was in the vicinity of a city nursery garden, which could have been its entry point. Although the population of E. vulnerata was found in vegetation near the customs area in Belgrade, this fi nding does not necessarily imply that it was introduced by trading and transport.
Unlike the numerous and seemingly welladjusted population of A. conica observed in Novi Sad, it is diffi cult to evaluate the accommodation level of O. ishidae and E. vulnerata due to their fairly low number of detected individuals. All three species, along with Ph. cyclops, are more or less Vitisrelated, whether as disease vectors or physical damage inducers (Zimmerman et al., 1996;Lessio et al., 2016;Duso et al., 2019;Plavec et al., 2019). Even though O. ishidae was not detected in vineyards, and only one Ph. cyclops specimen was collected on grapevine, agroeconomic losses that accompanied the emergence of S. titanus and FD in Europe should be considered when assessing the fi ndings of these two leafh oppers in Serbia. Assumptions regarding plant associations of the polyphagous O. ishidae in Serbia would be very speculative. Th e fi ndings of solitary nymphs on P. alba that almost certainly belong to O. ishidae, suggest that this plant could be preferred in Serbia as it is in Italy (Parise, 2017). A single specimen of Ph. cyclops was swept from Ulmus sp. in central Serbia, which would be   (Chen et al., 2011). Th is data should be taken with caution, given that the photograph of the specimen noted as "Ph. cyclops" in that paper is misleading (de Haas, M., personal communication). Th erefore, examination of male genitalia should be essential for the genus Phlogotettix, as well as for other Auchenorrhyncha species in order to avoid vague data (Meshram et al., 2015). Th e North American species A. conica and E. vulnerata are not known as plant disease vectors, but being associated with grapevine, they aff ect the plant's fi tness. Interestingly, interactions between E. vulnerata and European grapevine cultivars were fi rst investigated in the USA (Zimmerman et al., 1996). Th e fi rst occurrence of this species in Europe was followed by leaf curling, discoloration and scorching of V. vinifera and V. lambrusca, which hosted insect populations (Duso et al., 2005). Grapevine plants from which E. vulnerata was collected in Belgrade did not show any of the aforementioned symptoms, which can be due to low abundance of the observed population or the phenological period of specimens transitioning toward their overwintering sites. Th e biology of polyphagous A. conica is very similar to the life traits of M. pruinosa, known to cause severe damage of wild and cultivated plants by producing large amounts of wax and honeydew, and enhancing the development of mildew and leading to fruit decay (D'Urso & Uliana, 2006). Although A. conica has not yet been recorded on grapevine in Serbia, progressing out of the urban area of Novi Sad may lead this species towards the vine-growing region of Fruška Gora in the vicinity.
First fi ndings of O. ishidae, A. conica and E. vulnerata, and confi rmed presence of Ph. cyclops, come from urban and natural ecosystems, indicating their phenological independence from cultivated plants and crops. Nevertheless, all four species should be accounted for in the context of grapevine protection since their intrusion into vineyards can be erroneous event or can lead to the establishment of populations inside crops which may cause plant physical damage and/or induce phytoplasma-borne disea ses. We strongly encourage future research of this insect group to focus on interactions that Auchenorrhyncha species establish within natural ecosystems because invasive potential can be predicted only as a result of comprehensive knowledge of the species life traits.