Introduction
According to the results of the Delivering Alien Invasive Species Inventory for Europe project (Roques et al. Reference Roques, Rabitsch, Rasplus, Lopez-Vaamonde, Nentwig and Kenis2009), alien insects and other terrestrial arthropods are the most numerous invaders in Europe, manifesting various effects, particularly in agricultural and forest ecosystems. Impacts range from the negative environmental effects commonly combined with economic losses, to negative social impacts when they affect a human well being (Binimelis et al. Reference Binimelis, Born, Monterroso and Rodríguez-Labajos2007). Once established into a new environment, alien terrestrial arthropods, especially insects, may undergo rapid dispersal followed by exponential population growth due to a lack of natural enemies that regulate their populations in their native areas, also referred to as enemy release hypothesis (Keane and Crawley Reference Keane and Crawley2002).
According to Roques et al. (Reference Roques, Rabitsch, Rasplus, Lopez-Vaamonde, Nentwig and Kenis2009), a total of 1296 alien terrestrial invertebrates originating from other continents are documented to have established in Europe; of which arthropods, mostly insects, represent 94% of the introduced species. The number of introduced species is probably even higher, taking into account the fact that identification and recognition of newly introduced alien species is often delayed until populations of introduced insects become well established and begin producing distinct environmental or economic impacts. Lack of taxonomic expertise in determination of the alien species may cause delays in recognition of the taxa, especially with regards to native and/or previously introduced congeneric species with high morphological similarity.
Over the past 50 years, several alien species in the suborder Auchenorrhyncha (Hemiptera) originating from the Nearctic Region caused various negative impacts in Europe by inducing substantial economic losses and environmental disturbance (Arzone et al. Reference Arzone, Vidano and Alma1987; Foissac and Wilson Reference Foissac and Wilson2009). The most striking example is a North American leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae), which was first reported in 1958 in the vineyards of southwestern France (Bonfils and Schvester Reference Bonfils and Schvester1960). The species quickly spread to southeastern France (Schvester et al. Reference Schvester, Carle and Moutous1963), northwestern Italy (Vidano Reference Vidano1964), and southern Switzerland (Baggiolini et al. Reference Baggiolini, Canevascini, Caccia, Tencalla and Sobrio1968). Presently, S. titanus colonises vineyards in all Mediterranean countries including the temperate regions of Austria and southeastern Europe. Introduced from eastern parts of North America to Europe (Papura et al. Reference Papura, Burban, Van Helden, Giresse, Nusillard and Guillemaud2012), this leafhopper became a causal vector in transmission of a quarantine disease, Grapevine flavescence dorée (Bacteria: Acholeplasmataceae) (Caudwell et al. Reference Caudwell, Kuszala, Bachelier and Larrue1970), caused by a phytoplasma belonging to the 16SrV taxonomic group (Angelini et al. Reference Angelini, Squizzato, Lucchetta and Borgo2004; Arnaud et al. Reference Arnaud, Malembic-Maher, Salar, Bonnet, Maixner and Marcone2007). It is worth noting that species of the genus Scaphoideus Uhler are also considered as potential vectors in transmitting diverse phytoplasma diseases in North America (Hill and Sinclair Reference Hill and Sinclair2000; Beanland et al. Reference Beanland, Noble and Wolf2006). In several large and small outbreaks from 1970 to 2006, Grapevine flavescence dorée severely affected vineyards in France, Spain, Portugal, Italy, and Serbia causing major economic losses due to deleterious effects on grapevine and the costs of control measures against the vector and disease spread.
Another well-known exotic species is the planthopper Metcalfa pruinosa (Say) (Hemiptera: Flatidae), also introduced from North America into the Mediterranean and temperate regions of Europe (Zangheri and Donadini Reference Zangheri and Donadini1980). Besides the great invasiveness of this planthopper in Europe, its presence is associated with serious damage to various field crops, gardens, and ornamental plants. The gregarious feeding behaviour of its nymphs and the massive secretion of epicuticular wax and honeydew favour the development of fungal diseases in the affected plants (Byrne and Bellows Reference Byrne and Bellows1991).
In the present paper, we document for the first time a species of the genus Osbornellus Ball (Hemiptera: Cicadellidae) identified as Osbornellus auronitens (Provancher) introduced to the Palaearctic from the Nearctic region based on morphological and molecular evidence. We report morphological features to distinguish it from the most closely related species in Europe, S. titanus. Finally, we discuss the possible implications of its expected spread across the Old World and possible interactions with native plants in its new environment.
Materials and methods
Insect sampling
During extensive study of Auchenorhyncha fauna in a wine-growing region in Canton Ticino (Southern Switzerland), a total of three males of a leafhopper species resembling Scaphoideus were collected on 11 August 2016 in a woody patch scattered within a wine-growing area (Stabio, 45°51'17.2'N, 8°55'38.1'E). At the collection site, Castanea sativa Miller (Fagaceae), Corylus avelana Linnaeus (Betulaceae), and Alnus glutinosa (Linnaeus) Gaertner (Betulaceae) are the dominant trees intermixed with scarce Cornus sanguinea Linnaeus (Cornaceae), Salix Linnaeus (Salicaceae), and Rubus Linnaeus (Rosaceae). The site was revisited on 7 and 9 September, and three more specimens (two females and one male) belonging to the same species were collected. All specimens were collected using a sweep net and mouth aspirators.
Morphological study
The abdomen of a voucher specimen designated as Oa1 (male) was dissected to study the genitalia (Knight Reference Knight1965), thereafter identified by using published taxonomic keys and related literature (Beamer Reference Beamer1937; DeLong and Knull Reference DeLong and Knull1941; DeLong Reference DeLong1941a; DeLong and Martinson Reference DeLong and Martinson1976a, Reference DeLong and Martinson1976b; Dlabola Reference Dlabola1984, Reference Dlabola1987a, Reference Dlabola1987b). Photographs were taken with a Leica MC 170AD digital camera on the Leica M165C stereomicroscope (Wetzlar, Germany). Terminology follows Viraktamath and Mohan (Reference Viraktamath and Mohan2004). Voucher specimen Oa1 is deposited in the collection of the Natural History Museum in Lugano (Canton Ticino, Switzerland). The rest of the sampled material is deposited in the private collection of the first author.
Molecular analysis
For molecular analysis, DNA was extracted from a voucher specimen designated as Oa2. The barcoding region of mitochondrial cytochrome oxidase subunit I gene (mtCOI) was used to confirm the identity of the sampled leafhoppers. Genomic DNA was extracted using the DNeasy® Blood & Tissue Kit (Qiagen Inc., Valencia, California, United States of America) following the manufacturer’s instructions. The barcoding region of the mtCOI gene was amplified using the primer pair LCO1490/HCO2198 (Folmer et al. Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994). Each polymerase chain reaction was carried out in a volume of 20 μL [1 μL of DNA, 11.8 μL of H2O, 2 μL of high yield reaction buffer A with 1x Mg, 1.8 μL of MgCl2 (2.25 mM), 1.2 μL of dNTP (0.6 mM), 1 μL of each primer of the pair LCO1490/HCO2198 (0.5 μM), and 0.2 μL of KAPATaq DNA polymerase (0.05 U/μL) (Kapa Biosystems, Wilmington, Massachusetts, United States of America)]. The polymerase chain reaction protocol consisted of an initial denaturation at 95 °C for five minutes, 35 cycles each consisting of three steps, i.e., one minute at 94 °C, one minute at 54 °C, and 1.5 minute at 72 °C, with a final extension step at 72 °C for seven minutes at the end of amplification protocol. Amplified products were run on 1% agarose gel, stained with ethidium bromide and visualised under an ultraviolet transiluminator. Polymerase chain reaction amplicons were sequenced by Macrogen (Seoul, Korea). To obtain 658 base pairs of the barcoding region sequencing was performed with both, forward and reverse primers. Sequence data are deposited in GenBank (www.ncbi.nlm.nih.gov) under accession number KY006567.
Results
According to morphology and molecular analysis, the specimens collected in Stabio (Canton Ticino, Switzerland) were identified as O. auronitens. The barcoding sequence of mtCOI subjected to BLAST analysis (www.ncbi.nlm.nih.gov/BLAST) revealed 100% identity with a sequence of O. auronitens (accession number KR043272), originating from Churchill, Prince Edward Island, Canada (Gwiazdowski et al. Reference Gwiazdowski, Foottit, Maw and Hebert2015).
Osbornellus auronitens (Provancher)
Material examined
Four males (designated as voucher specimens Oa1–Oa4), Stabio, Ticino, Switzerland (45°51'17.2'N, 8°55'38.1'E), 11.viii.2016 and 9.ix.2016, V. Trivellone; two females (Oa5, Oa6) Stabio, Ticino, Switzerland, 7.ix.2016 and 9.ix.2016, V. Trivellone. Voucher specimens Oa1 is deposited in the Natural History Museum in Lugano, while voucher specimens Oa2–Oa6 are in the private collection of V.T.
Differential diagnosis
Osbornellus auronitens is morphologically similar to S. titanus, a well-known vector of phytoplasmas belonging to the elm yellows ribosomal group and introduced into the Palaearctic region in the 1960s. The overall size, shape, and colouration bear a striking resemblance between the two species, but they can be distinguished using distinctive external morphological characters.
In O. auronitens, the anterior margin of the head has two dark brown, transverse bands; an incomplete brown preapical band on the vertex; and an arcuate, red-orange fascia between the eyes more posterad (Fig. 1A–B). In S. titanus, the head has four transverse, brown bands – two on the upper part of the face below the ocelli and two on the vertex, and a transverse light-orange fascia between the eyes with a median angulate projection anteriorly (Fig. 1D–E). The forewings of the two species are similar in colouration and venation but O. auronitens has relatively few dark brown markings and vein R has only three branches (Fig. 1A–B) while S. titanus has the dark markings more extensive and vein R has five branches (Fig. 1D–E). As in other species of Osbornellus, the male subgenital plate of O. auronitens has an elongate, slender, filamentous distal portion (Fig. 1C). In contrast, the subgenital plate of S. titanus is broadly rounded distally and lacks an elongated distal portion (Fig. 1F). All of the above traits are visible without dissection.
Fig. 1 Habitus of Osbornellus auronitens (Provancher), voucher specimen Oa1, A–C and Scaphoideus titanus Ball from Serbia (Bela Crkva, Banat region), D–F in dorsal, lateral view and ventral view respectively.
In Fig. 2, dissected parts of the male genitalia of O. auronitens are shown. The aedeagus of O. auronitens in lateral view is slender and thin, slightly curved dorsally, with a pair of broad processes arising laterally near the base of the shaft, flaring slightly, extending just beyond the apex of the shaft to dorsally pointed apices (Fig. 2A–C). In S. titanus, the aedeagus in lateral aspect is generally triangular with the base generally pear shaped; the paired posterior processes arising from the connective stem are membranous, digitiform, and divergent in ventral view, usually not reaching the apex of the aedeagus (della Giustina Reference della Giustina1989). The style in O. auronitens has the distal section stout, parallel sided, and relatively long, and the apex rounded and slightly concave on laterally near the apex (Fig. 2G–I). In S. titanus, the distal portion of the style is distinctly tapered and curved laterally with two setae near the middle and the apex is sharply pointed (della Giustina Reference della Giustina1989). The shape of the male pygofers of O. auronitens and S. titanus are rather similar.
Fig. 2 Male genitalia of Osbornellus auronitens (Provancher) voucher specimen Oa1. A, Aedeagus, dorsal view; B, aedeagus, rotate 30°; C, aedeagus, lateral view; D, left subgential plate, dorsal view; E, left subgential plate, lateral view; F, valve and right subgential plate; G, left stylus, dorsal view; H, left stylus, ventral view; I, left stylus, lateral view; J, pygofer, dorsal view.
Discussion
Early detection of an alien organism is of crucial importance to allow observation of the vast array of possible interactions between the introduced organism and its new environment. Developing a monitoring strategy is also important for minimising possible consequences of such an introduction. Most leafhopper species are small, inconspicuous animals that when introduced into new areas, may remain unnoticed for some time. Early detection may also be hindered in cases, such as the one presented here, when a newly introduced species is morphologically similar to another species already present. Unfortunately, due to a lack of systematic survey and monitoring programmes designed to detect exotic species, the presence of such species is most often noticed as a result of their negative environmental or economic impacts after they have become well established.
Although O. auronitens is morphologically similar to S. titanus, as noted above, in Europe it is possible to distinguish these species reliably using external morphological traits. So far, only one other species of Osbornellus, O. horvathi (Matsumura) (Hemiptera: Cicadellidae), and one other species of Scaphoideus, S. dellagiustinai Webb and Viraktamath (Hemiptera: Cicadellidae), have been reported from western Europe. Osbornellus horvathi, which is reported from Algeria, Sicily, and Spain, is similar to S. titanus in external colour pattern but easily distinguishable by the structure of the male genitalia (Hermoso de Mendoza et al. Reference Hermoso de Mendoza, Del Estal, Alcázar, Pérez-Otero and Marsilla2012). This species has recently been shown to be capable of transmitting ‘Candidatus Phytoplasma asteris’ to broad bean (Vicia faba Linnaeus; Fabaceae) and periwinkle (Apocynaceae) in experimental trials (Rizza et al. Reference Rizza, Pesce, D’Urso, Raciti, Marzachì and Tessitori2016). Scaphoideus dellagiustinai, which is, so far, only recorded from the coast of southern France and Burkina Faso, is smaller and paler in colouration compared with both O. auronitens and S. titanus; has only two transverse, brown bands on the head, both on the face below the ocelli; the vertex is white with a single, bright red, transverse band; and the male genitalia are distinctive (see figures in Webb and Viraktamath Reference Webb and Viraktamath2007).
Within their native range of eastern North America, both O. auronitens and S. titanus species can be confused with congeneric species occurring in the same deciduous forest habitats that are similar in size, colour pattern, and wing venation. Thus, it is important to confirm the identities of these species by examining features of the male genitalia that are diagnostic at the species level. Osbornellus and Scaphoideus are large genera, each with over 100 documented species worldwide and it is quite possible that additional species of both genera could eventually become established in Europe.
The genus Osbornellus is presently included in the tribe Scaphoideini within the large cicadellid subfamily Deltocephalinae. The genus was originally established by Ball (Reference Ball1932) based on type species Scaphoideus auronitens Provancher. Ball’s (Reference Ball1932) decision to recognise additional genera in the group was based on observations of morphological differences and distinct feeding behaviour and ecological relationships. The genera were originally distinguished based on the size of the ocelli and characteristics of the veins and cells of the forewing. Beamer (Reference Beamer1937) revised Osbornellus in the United States of America, highlighting the difficulty of distinguishing species. Subsequent authors added nearly 100 species from the Nearctic and Neotropical regions (e.g., DeLong and Berry Reference DeLong and Berry1937; DeLong Reference DeLong1941a, Reference DeLong1941b, Reference DeLong1942, Reference DeLong1976; DeLong and Knull Reference DeLong and Knull1941; Metcalf Reference Metcalf1954; Linnavuori Reference Linnavuori1959; DeLong and Martinson Reference DeLong and Martinson1976a, Reference DeLong and Martinson1976b) mainly distinguished using features of the male genitalia. Martinson (Reference Martinson1977) completed a taxonomic revision of the genus as a doctoral dissertation, but this work was never published. Dominguez and Godoy (Reference Dominguez and Godoy2010) recently added 37 new Osbornellus species from Central America.
Nine species of Osbornellus have been recorded from the Palaearctic, four of which have been assigned to the endemic western Asian subgenus Mavromoustaca Dlabola and one to the nominotypical subgenus (Matsumura Reference Matsumura1908; Vilbaste Reference Vilbaste1976; Dlabola Reference Dlabola1984, Reference Dlabola1987a, Reference Dlabola1987b). However, the phylogenetic status of the genus, and relationships of subgenera and species presently included in the genus, has never been explored through phylogenetic analyses (Nielson and Knight Reference Nielson and Knight2000).
Within its native range, O. auronitens is widespread in the deciduous forests of eastern North America from Canada (southern Ontario and Québec) to the southeastern United States of America (Florida to Oklahoma) (Martinson Reference Martinson1977). Its introduction into the Palaearctic Region may have occurred through contamination of imported live plant material (Bertin et al. Reference Bertin, Guglielmino, Karam, Gomulski, Malacrida and Gasperi2007), especially because O. auronitens hibernates as eggs inserted into the bark of woody plants. This is also the case with mosaic leafhopper Orientus ishidae (Matsumura) (Hemiptera: Cicadellidae), a polyphagous species native to the eastern Palaearctic Region but also widespread and well established in eastern North America (Valley and Wheeler Reference Valley and Wheeler1985), and reported for the first time in Europe from Switzerland in 2002 (Günthart and Mühlethaler Reference Günthart and Mühlethaler2002). Over the subsequent 15 years, the mosaic leafhopper was recorded in Germany (Nickel and Remane Reference Nickel and Remane2003), Slovenia (Seljak Reference Seljak2004), Czech Republic (Malenovský and Lauterer Reference Malenovský and Lauterer2010), Austria (Nickel Reference Nickel2010), France (Mifsud et al. Reference Mifsud, Cocquempot, Mühlethaler, Wilson and Streito2010), Hungary (Koczor et al. Reference Koczor, Bagarus, Karap, Varga and Orosz2013), and Serbia (V.T., unpublished data). Several authors also suggested that O. ishidae had probably been introduced into Europe by trading of plants (Malenovský and Lauterer Reference Malenovský and Lauterer2010; Mifsud et al. Reference Mifsud, Cocquempot, Mühlethaler, Wilson and Streito2010). The situation with O. ishidae is a typical case of rapid range expansion of the introduced alien leafhopper with possible economic impacts, especially after Grapevine flavescence dorée was identified recently in mosaic leafhopper adults from Italy, Slovenia, and Switzerland (Mehle et al. Reference Mehle, Seljak, Rupar, Ravnikar and Dermastia2010; Gaffuri et al. Reference Gaffuri, Sacchi and Cavagna2011; Trivellone et al. Reference Trivellone, Filippin, Narduzzi-Wicht and Angelini2016).
Data regarding capability of the species from the tribe Scaphoideini to serve as vectors in transmission of phytoplasmas are scarce in literature (Weintraub and Beanland Reference Weintraub and Beanland2006). Besides S. titanus, which is a documented vector transmitting the phytoplasma-associated disease Grapevine flavescence dorée on grapevine in Europe, O. auronitens was noted as a possible vector involved in transmission of grapevine yellows diseases in vineyards of Virginia, United States of America (Beanland et al. Reference Beanland, Noble and Wolf2006).
Although it is not known whether O. auronitens will disperse into new areas in the Palaearctic, almost 60 years ago, S. titanus, a species with similar food preferences in its native range, was introduced in France and is now widespread from 35 to 48 degrees north latitude and from Portugal to Romania. Scaphoideus titanus adjusted well to its new European environment, and has colonised both wild and cultivated grapevine to become the major vector of phytoplasma causing the Flavescence dorée disease on grapevine. Osbornellus auronitens collected in Switzerland has colonised the ecotone strip between vineyards and forest. This suggests that it poses a potential risk, as already reported from Virginia, where S. titanus, O. auronitens, and Jikradia olitorius (Say) (Hemiptera: Cicadellidae) exhibit seasonal movement into vineyards from nearby forest vegetation, which could account for the high incidence of diseased vines observed near the vineyard edge (Beanland et al. Reference Beanland, Noble and Wolf2006). Thus, presence of O. auronitens and its potential spread across viticultural regions in Europe is of objective concern because polyphagous adults can acquire different phytoplasmas from adjacent plant vegetation and launch new epidemiological cycles inside vineyards. To prevent such risk, setting controlled conditions for import of all plant material from foreign countries is of utmost importance. Scientific and economic concern that the spread of this exotic leafhopper species could lead to tremendous economic losses is justifiable because a similar scenario has already occurred in Europe with introduction and establishment of the related species, S. titanus.
Acknowledgements
The authors thank the Swiss National Science Foundation for financial support of this study through the Scientific co-operation between Eastern Europe and Switzerland (SCOPES) programme (grant number IZ73Z0_152414).
