Turkish Journal of Zoology Turk J Zool (2019) 43: 531-535 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Short Communication doi:10.3906/zoo-1905-6

Molecular identification of invasive drosophilidZaprionus tuberculatus Malloch (Diptera: ) in Çanakkale, Turkey*

Akın KUYULU, Seda YÜCEL, Hanife GENÇ** Department of Agricultural Biotechnology, Faculty of Agriculture, Çanakkale Onsekiz Mart University, Çanakkale, Turkey

Received: 07.05.2019 Accepted/Published Online: 10.07.2019 Final Version: 02.09.2019

Abstract: The vinegar or pomace , tuberculatus Malloch, is native to the Afrotropical region but has recently colonized new areas in Turkey, Italy, Malta, Romania, and Greece. This invasive species was first detected in 2012 in Adana on banana and peach baits and is considered a potential agricultural pest in fig production in Turkey. However, the European and Mediterranean Plant Organization reports that no direct association of the fly with fruit damage has been documented so far because specimens were generally caught in traps. Specimens of Zaprionus sp. were monitored during surveys for cases of suzukii (Matsumura) and Ceratitis capitata (Wiedemann) damaging experimental pomegranate and fig fields in Çanakkale, where we performed the field study. To better understand the species identification, a partial sequence of the mitochondrial cytochrome oxidase I COI( ) gene region was used in this study. The barcode primer pairs were used to amplify the partial COI gene. PCR products were visualized and then directly sequenced. No insertion or deletion was observed in the 637-bp COI gene region. The average nucleotide frequencies of T, A, C, and G were observed as 39.1%, 29.6%, 15.2%, and 16.1%, respectively. Intraspecific pairwise divergence was low at 0.15%. Nucleotide sequences indicated that there were 2 substitutions, an A→G at 343 and a C→T at 592 positions, respectively. In conclusion, this study presents the first molecular evidence and presence ofZ. tuberculatus in Çanakkale, Turkey, which can help in resolving morphologic ambiguities.

Key words: , molecular identification,COI , mtDNA, Çanakkale

The genusZaprionus Coquillett, 1902 is known int/ACTIVITIES/plant_quarantine/alert_list_insects/ to have 2 subgenera. Zaprionus sensu stricto, with 46 zaprionus_tuberculatus) and was recently found in Greece species, originated from the Afrotropical region. The other (Crete), feeding on mature fig fruits during the summer subgenus, called Anaprionus, with 11 species, is native to of 2017 (Leivadaras et al., 2017). Zaprionus tuberculatus is the Oriental and Australasian regions (Yassin, 2008; Yassin morphologically similar to Z. sepsoides, Z. mascariensis, Z. and David, 2010). The is abundant in (Tsacas kolodkinae, and Z. verruca; all specimens have a tubercule et al., 1981) but is now also known to be present in the on the fore femur and are considered as sibling species Palearctic region (Yassin, 2008). The Zaprionus species (Tsacas et al. 1981; Yassin, 2008). differ from each other based on testes size and sperm A few drosophilid species are known to be primary ultrastructures, and they are also phylogenetically close to pests, such as Drosophila suzukii Matsumura (Walsh et the genus Drosophila (Commar et al., 2012). al., 2011) and Gupta (Lasa and Tadeo, Zaprionus tuberculatus Malloch was first described 2015; Kremmer et al., 2017); however, Z. tuberculatus seems in 1932 in South Africa. The common name is vinegar to be a potential pest based on recent reports (Leivadaras fly or pomace fly and it originated in tropical Africa et al., 2017). It has been newly introduced as an invasive (Yassin, 2008). It is present in the eastern African islands drosophilid in Turkey and was first detected in 2012 in of Mauritius and Madagascar. It was also suggested Adana (Patlar et al., 2012), based on its morphological that it should be added to the alert list of the European identification key characters (Yassin and David, 2010). and Mediterranean Plant Organization in 2017. It has Since then, this pest has received little attention and, even more than 49 tropical fruit hosts (https://www.eppo. though it was caught in traps, there have been no reports * Part of this research was presented as a poster at the 1st International Symposium on Biodiversity Research in Çanakkale, Turkey (2–4 May 2019) and published as an abstract in the abstracts book. ** Correspondence: [email protected] 531

This work is licensed under a Creative Commons Attribution 4.0 International License. KUYULU et al. / Turk J Zool about its distribution, host range, and pest status in Turkey °C, 1 min at 48 °C, 45 s at 72 °C, and 10 min at 72 °C. (Patlar et al., 2012). Amplicons were confirmed on 1% agarose gel and then We surveyed orchards in Çanakkale Province for the sent to a company for sequencing. The resulting sequences presence of D. suzukii and Ceratitis capitata to improve were compared in the NCBI database (GenBank) using our laboratory population with the addition of wild . BLASTn with standard settings (Altschul et al., 1990) and We monitored the damage on overripe fruit by hand and deposited in GenBank (Accession Numbers MK509763– air aspirator and brought the specimens to the laboratory MK509766). for further identification. We then caught several adults Sequences were checked manually to avoid misreading, belonging to the genus Zaprionus based on morphological and MEGA-X software was used for analysis (Kumar et characters, but external morphological details of the al., 2018) followed by alignment using Clustal W (1.6) species belonging to genus Zaprionus are so similar that to with default parameters; assembled sequences were then be certain about the species level molecular identification collapsed into haplotypes. The sequenced haplotypes were was performed. checked for no internal stop codons to confirm original Mitochondrial DNA markers have been used to identify mtDNA sequences (Zhang and Hewitt, 1996; Rokas et al., and discover the origin of pest invasions in previous 2003; Mutun and Atay, 2015). studies (Yassin et al., 2010; Frank et al., 2011). Because the Phylogenetic trees were built using the maximum specimens were so tiny and normally need to be identified parsimony (MP) method with bootstrap values based on by a specialist, we used molecular tools to determine and 1000 replications to assess branch support (Felsenstein, correlate morphologically identified specimens. In the 1985). MP analysis was performed using the tree- present study, we report on the first observation of an bisection-regrafting (TBR) algorithm with search level invasive population of Z. tuberculatus in Çanakkale. 1, where the previous trees were received by randomly Samples of Z. tuberculatus were monitored during adding sequences (10 replicates) (Nei and Kumar, 2000). surveys of damage by D. suzukii and Ceratitis capitata to Z. tuberculatus was recently introduced to the fig and pomegranate experimental fields on the Dardanos Palearctic region and monitored in D. suzukii field traps Campus of Çanakkale Onsekiz Mart University in October (Lasa and Tadeo, 2015; Kremmer et al., 2017). Pest status 2018. The flies were collected from infested pomegranates and other biological parameters are important to evaluate and figs with an air aspirator and brought to the laboratory. its potential damage on healthy fruit. Field-collected flies About 20 to 25 flies were placed in a plastic cup (30 × 18 were reared on pears, strawberries, figs, and pomegranates, × 8 cm) along with a piece of fig fruit Ficus( carica L.) which were all ripe but undamaged fruit in this study. The and covered with a chiffon lid for egg-laying to establish females laid numerous eggs in groups in all tested healthy a laboratory colony. Strawberry (Fragaria ananassa), fruit (Figures 1A and 1B). The eggs have 4 long filaments, pear (Pyrus sp. L.), and pomegranate (Punica granatum) characteristic to African Zaprionus s.s. (Yassin and David, were also used to colonize flies in the laboratory. The 2010); on the other hand, the genus Drosophila has 2 remaining 4 flies were stored in 100% ethyl alcohol for filaments. It is possible to see both species’ eggs inside the further molecular analysis. Morphological identification same fruit but they are easily separated into 2 genera based of adult specimens was performed under a stereo zoom on the egg’s filaments. Larvae were able to feed on the soft microscope based on key characters (Yassin and David, tissue of the fruit and then pupated inside or outside of 2010). the fruit. Adults were easily distinguished, having reddish- For molecular analysis, DNA was isolated brown heads and unique silvery-white longitudinal strips individually (n = 4) from the whole body using a extending dorsally from the front of the head to the end GeneJET genomic DNA extraction kit following its of the scutellum (Figures 2A–2C). Another strip extends protocol. DNA content was quantified with a NanoDrop laterally from the thorax to the wing on both sides (Figure spectrophotometer. The barcode primer pairs LCO1490-F 2B), and the fore femur has a swollen tubercule with a (5´-GGTCAACAAATCATAAAGATATTGG-3´) and bristle, characteristic of Z. tuberculatus (Figure 2D). HCO2198-F (5´- TAAACTTCAGGGTGACCAAAAAAT The 4 specimens were first identified morphologically C-3´) (Folmer et al., 1994) were used to amplify the (Yassin and David, 2010) and then confirmed further mtDNA cytochrome oxidase I (COI) gene. Polymerase based on the results of mitochondrial sequences of the chain reaction (PCR) was conducted in a 25-µL final partial COI gene. PCR products yielded a 708-bp amplicon reaction volume including 2.5 µL of 10X buffer, 1 µL of for each individual, and they were successfully sequenced.

MgCl2 (25 mM), 0.5 µL of dNTPs (10 mM), 0.5 µL of After the sequences were trimmed, we used 637 bp of the each primer (10 pmol), 1 µL of template DNA, and 1 U of partial COI gene from 4 individuals for molecular analyses. Taq polymerase (Fermentas, USA). PCR conditions were The sequences were checked and did not contain any set as follows: 5 min at 94 °C; then 36 cycles of 40 s at 94 insertion, deletion, or stop codon in the partial COI gene.

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Figure 1. View of Zaprionus tuberculatus eggs laid in the soft tissues of a pear (A) and a close-up egg (B).

Figure 2. Zaprionus tuberculatus specimen. Dorsal view with unique silvery-white stripes (A); lateral view showing shorter stripe (B); adult laying eggs (C); fore femur with swollen tuberculus (D).

The average nucleotide frequencies of T, A, C, and G were at an accuracy of 100%. The best BLAST search for the observed as 39.1%, 29.6%, 15.2%, and 16.1%, respectively. H2 haplotype (Turkey 1-2-3 sequences) was obtained The base composition ofZ. tuberculatus sequences was with the sequences gb|FJ948782.1|, gb|EU493691.1|, and observed as the strong AT bias (68.7%) found frequently gb|EU493688.1|, which corresponded respectively to Z. in the mitochondrial genome of (Kremmer et al., verruca, Z. badyi, and Z. tuberculatus at an accuracy of 2017). 100%, as well. The 4 nucleotide sequences were then used The 4 nucleotide sequences indicated 2 haplotypes, further to construct a phylogenetic tree to show molecular which were different from each other by 2 nucleotide clustering among the genus Zaprionus and to confirm substitutions at 343 (A→G) and 592 (C→T) positions. species identification (Figure 3). The best BLAST search for the H1 haplotype (Turkey 1 The Afrotropical species of the genusZaprionus and sequence) was obtained with the sequence gb|LN867073.1|, subgenus Z. tuberculatus were revised and described which corresponded to Z. tuberculatus, an Italian specimen, again based on morphometrical and molecular

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Figure 3. Phylogenetic tree of Z. tuberculatus specimens collected from Çanakkale, Turkey. The MP tree is based on partialCOI sequences. Meigen and Drosophila suzukii Matsumura were used as outgroups.

534 KUYULU et al. / Turk J Zool techniques (Yassin and David, 2010). The subgenus The invasive Z. tuberculatus was first detected in 2012 Zaprionus is described as generalist and usually develops in Adana (southern Turkey) and currently has a colonized on decaying fruits (Patlar et al., 2012). Z. tuberculatus population in Çanakkale (northwest Turkey), so we has 49 different fruit species as larval hosts (Lachaise provided additional features that would help to confirm the and Tsacas, 1983). Z. tuberculatus could be a secondary specimens. Previous studies have reported that the genus pest feeding on overripe fruits in Çanakkale orchards Zaprionus is tropical and not expected to be established and seems to have recently established a population in colder climates (Araripe et al., 2004). However, recent there. Several scientific reports have pointed out its studies (Raspi et al., 2014; Kremmer et al., 2017) together recent invasions in Europe (Patlar et al., 2012; Raspi et with our observations show that changes in climatic al., 2014; Leivadaras et al., 2017). Indeed, its association conditions make the establishment of tropical species with D. suzukii in nature and its feeding on healthy fruits and new pest invasions possible and favorable. However, in the laboratory could contribute to damage in fruit further studies are still required to determine whether Z. crops. It will be important to monitor its pest status and tuberculatus is a primary pest or not and to monitor the its natural enemies. A closely related species, Zaprionus potential risk for commercial agricultural crops. indianus, is not considered a pest in native areas but infests undamaged fig fruits, having caused serious Acknowledgment damage in Brazil (Joshi et al., 2014) and also recently in The authors would like to thank James L. Nation Sr. for his France (Kremmer et al., 2017) useful suggestions on an earlier version of this manuscript.

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