The ongoing dispersion of the Eucalyptus bronze bug (Thaumastocoris peregrinus) in Spain

Aim of study: Thaumastocoris peregrinus (Carpintero & Dellapé, 2006) is notable for its dispersion potential, and for its damage to a wide range of hosts of the genus Eucalyptus. The intense movement of people and cargo between continents contributes to the success of its geographical distribution on the globe and hinders the adoption of preventive measures. The celerity and precision in the identification of T. peregrinus, as well as its invasion and dispersion routes are fundamental for the implementation of measures to prevent new invasions. Area of Study: Park of Retiro in the community of Madrid, Spain, where T. peregrinus is present. Material and methods: We analyzed a fragment of COI mtDNA gene in T. peregrinus specimens, using samples collected at a public park in Madrid urban area, to study the possible pathways of incursion of this insect in Spain. The goal was achieved using molecular tools, with PCR amplification of partial mtDNA COI and sequencing the fragment, which is used as a barcode of life for identification at species level. Species identity was confirmed using the database in GenBank. Results: The results confirms that the specimens found in Madrid are T. peregrinus, and all are from the same maternal lineage. Research highlights: We present the first molecular information of T. peregrinus population present in Spain, and suggest and discuss possible routes of incursion of this pest. Additional keywords: Invasive pest; DNA Barcoding; Invasion Routes; Forest Entomology. Authors ́ contributions: Conceived and designed the work: DNM, ECC, CRP, JAA, ACR. Performed the experiments and analyzed the data: DNM, ACR, CRP, JVL, MAS, GAU. Contributed materials/analysis tools: CRP, GAU, TLC. All authors assisted in writing and reviewed the article. Citation: Nascimento-Machado, D.-do, Corrêa-Costa, E., Perini, C.-R., Andrade-Ugalde, G., Alves-Saldanha, M., Leitão, J.-V., Lovato-Colpo, T., Arnemman, J.-A., Cordero-Rivera, A. (2019). The ongoing dispersion of the Eucalyptus bronze bug (Thaumastocoris peregrinus) in Spain. Forest Systems, Volume 28, Issue 3, eSC03. https://doi.org/10.5424/fs/2019283-15353 Received: 24 Jun 2019. Accepted: 31 Oct 2019. Copyright © 2019 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC-by 4.0) License. Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Dayanna do Nascimento-Machado: dayanasmac@gmail.com Funding agencies/Institutions Project / Grant Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil Scholarship to the first author in the Sandwich Doctorate Program Abroad 88881.187250/2018-01 Spanish Ministry of Science, Innovation and Universities CGL-2018-096656-B-100, including FEDER funds.

Based on the potential global distribution of the Bronze bug, European countries present localities with optimal climate conditions to its occurrence and dispersion (Saavedra et al., 2015a). However, there is no information about the genetic constitution of populations of T. peregrinus in Spain, even if this information is extremely important to know the diversity and possible routes of invasion, which are determined by the genetic signatures of the populations present on site. In this context, this work aims to report the expansion of T. peregrinus occurrence area in Spain and to characterize, using molecular analysis, the genetic structure and possible invasion routes of the population found in Madrid, Spain.

Specimens sampling
Bronze bug specimens were sampled around Spain from November 2018 to April 2019, in Galiza region, including Vigo, Pontevedra, Santiago de Compostela and Baiona, and also in Barcelona and in Madrid. Galiza region have optimal climate conditions to a potential distribution of T. peregrinus in Europe as reported by Saavedra et al. (2015a), but we could not find any specimens in this area. Specimens of T. peregrinus were found in a city park "Parque de El Retiro" at Madrid, Spain (40°25'07.8"N -3°41'13.2"W) (  on leaves, many egg masses (Fig. 1b) and presence of nymphs and adults (Fig. 1c). Eucalyptus trees had a phytosanitary status of infestation by other insects, including G. brimblecombei. The weather during the day of sampling was between 5-19°C and had a clear sky (www.accuweather.com). We believe that the population of T. peregrinus sampled was in the beginning of infestation period, because we found many eggs and not many nymphs and adults on leaves.
Sampling procedure was performed on lower leaves of these trees collecting randomly the branches with bronze bug infestation. Insects were removed from leaves using a paint-brush and placed them in a plastic container (20 ml) with 96% alcohol, totalizing 10 specimens from each tree.
Samples collected in April were sent to the Laboratory of Forest Entomology -UFSM, Santa Maria, RS, Brazil, where the morphological characterization of the specimens was performed according to the morphological characters described by Carpintero & Dellapé (2006). The gDNA extraction and molecular characterization of the specimens were performed in the Laboratory of Integrated Pest Management (LabMIP-UFSM).
In August 5 of the same year with temperature around 35ºC the trees were rechecked for infestation of bronze-bug and adults were found abundant, but eggs and nymphs were rare. Specimens from August were not sampled and not genetically analyzed, because the high probability of these insects to be family related.

DNA extraction, amplification and sequencing
Five insects were selected randomly from the specimens collected from each tree, for the extraction of gDNA. We used the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) to gDNA extraction of individual insects, according to the manufacturer's protocol. First of all, each adult was exposed to dry air in a paper towel in order to allow the ethanol evaporation. Then, we used 180 µL of buffer ATL and 20 µL of proteinase K to macerate the entire insect body in a 1.5 ml tube and left incubating at 56°C for 12 hours. Afterwards, gDNA was purified in a silicabased matrix and eluted in 35 µL of buffer AE. gDNA concentration was assessed using a NanoDrop™ 1000 (Thermo Scientific, Wilmington, DE, USA).
The PCR reaction was performed following Nadel et al. (2010) protocol: initial denaturation at 98°C for 30 s, followed by 30 cycles at 95°C for 30 s, 48°C for 30 s, and 72°C for 1.5 min, and a final extension at 72°C for 10 min. The amplified products of mtDNA (COI fragment) were resolved on 1.0% agarose electrophoresis gel, pre-stained with Nancy-520 DNA gel stain (Sigma-Aldrich) and visualized with a gel documentation system. PCR products that were successfully amplified were sequenced by ACTGene genomics service provider (Alvorada, RS, BR), using an AB 3500 Genetic Analyzer.

Data analysis
The data from sequencing output were trimmed, edited and analyzed for each individual COI sequence, using the software Pregap and Gap4 within the Staden package (Staden & Bonfield, 2000). CLC Sequence Viewer (Version 7.8.1 -QIAGEN Aarhus A/S) was used to cut the ends, retrieve and align sequences of 468 bp long, based on a sequence model from GenBank (FJ623760). In addition, a pBlast analysis (amino acid homology confirmation) with T. peregrinus partial mtDNA COI genes on NCBI (National Center for Biotechnology Information, USA database) was performed. The sequence of T. peregrinus from Madrid was deposited in GenBank under accession number MN401749.

Results
From the morphological and molecular characterization of the adult insects found in the Parque de El Retiro of Madrid, we confirm that all the sequenced specimens belong to the species Thaumastocoris peregrinus (Carpintero & Dellapé, 2006). All the specimens belong to the haplotype A, described by Nadel et al. (2010).

Discussion
The expansion of the area of T. peregrinus in Spain was already expected, since the intense traffic of people and goods (Hurley et al., 2016) and because the climatic suitable conditions predicted by CLIMEX, that reports between a low and moderate potential distribution of Dayanna do Nascimento-Machado, Ervandil Corrêa-Costa, Clérison Régis Perini, et al.

Forest Systems
December 2019 • Volume 28 • Issue 3 • eSC03 4 this pest on Madrid region (Saavedra et al., 2015a). Presently this species has been widely distributed in some countries, e.g. as Brazil  and Portugal (Garcia et al., 2013). Currently, it is known that only four strains of T. peregrinus of the eight haplotypes found by Nadel et al. (2010) and the 44 haplotypes found by Lo et al. (2019) in Australia, are scattered in different countries, two in South Africa (D and G haplotypes), one in South America (samples from Argentina, Brazil, Uruguay) (haplotype A -FJ623760) (Nadel et al., 2010) and one in Italy (IT haplotype -KF437485), not yet found in the center of origin of the pest (Nugnes et al., 2014).
The T. peregrinus population found in Madrid belongs to haplotype A, and there is no genetic diversity among the insects collected. Generally, in their area of natural occurrence, the species possess greater genetic diversity in relation to populations introduced in new environments (Puillandre et al., 2008).
When analyzing the first occurrence records of T. peregrinus in Europe, we verified that Italy (2011) (Laudonia & Sasso, 2012) and Portugal (2012) (Garcia et al., 2013) reported the presence of the insect before its detection in Spain. Therefore, we can suggest some hypothesis of introduction of T. peregrinus in Spain (Fig.  2), based on the genetic information of the population of Madrid. The most likely route of introduction may have been through the border with Portugal by natural dispersion (flight, hitchhiking in birds), transit of people and plant material or unintentionally by the importation of Eucalyptus timber from Latin America.
According to Garcia et al. (2013), most of the introductions of invasive alien species are mainly due to human activity, and T. peregrinus was probably introduced in Portugal through the importation of eucalypt logs from a Latin American country. In fact, the populations of T. peregrinus from Brazil, Argentina and Uruguay belong to the same haplotype found in Spain (Nadel et al., 2010). Thus, it is suggested that the haplotype present in Portugal is also haplotype A. Alternatively, it could have been introduced from Italy, however, the analysis of mtDNA from specimens of the population found in Italy have revealed the presence of a distinct haplotype, IT (Nugnes et al., 2014).
An introduction from Israel would be improbable because the first record of T. peregrinus was dated one month earlier than in Spain (Novoselsky & Freideberg, 2016). Finally, a direct introduction from Australia would also be very unlikely. The possible routes of introduction in Spain, can be confirmed when DNA analysis of populations of Portugal became available, in order to determine which lineage(s) of T. peregrinus are in the country. In addition, specimens from the region of Badajoz, Spain, could confirm the presence of the same haplotype, due to the proximity to the border of Portugal, where the pest is widespread (Garcia et al., 2013). We believe that an update of the genetic diversity of this species should be carried out in South America, Italy and South Africa, to find out if new introductory events with new haplotypes have occurred, thus increasing diversity in these places.
Thaumastocoris peregrinus is considered a prolific species, however, insects can go unnoticed until their abundance becomes critical. In Israel, it has been reported that these insects cause irritation and eruptions on the skin of some people who visited parks, possibly due to the direct contact of the specimens with the skin (Novoselsky & Freideberg, 2016). In this sense, the population of T. peregrinus found in a park in  Madrid, where the movement and the presence of people is intense, should be monitored. After detection, if preventive measures are not taken to control invasive species, their complete eradication will be more unlikely due to increasing population growth (Harvey & Mazzotti, 2014). It is important to highlight that eucalypt plantations in Spain comprise an area of approximately 500,000 ha (IFN, 2011;Santolamazza-Carbone et al., 2019).
There are no effective strategies for controlling T. peregrinus. Using pesticides, however, there was a significant reduction in populations in trees treated with imidacloprid in Australia (Noack et al., 2009). The results indicated that lambda-cyhalothrin + thiametoxam (1.2 + 25.8 g / ha) showed 100% control of insects after 24 hours of exposure (Machado et al., 2016). On the other hand, biological control is being widely diffused for T. peregrinus. Two species of Mymaridae wasps, Cleruchoides noackae Lin et Huber and Stethynium sp. Enock were characterized as parasitoids of T. peregrinus eggs in Australia (Lin et al., 2007). The species C. noackae was introduced in Brazil in 2012 and released in several states (Wilcken et al., 2015), because the introduction and successful establishment of natural enemies is important to regulate pest populations (Gerard et al., 2011;Thompson & Reddy, 2016). In addition, the species Chrysoperla externa (Hagen, 1861) (Neuroptera: Chrysopidae), was found preying nymphs of T. peregrinus  and Atopozelus opsimus (Elkins, 1954) (Hemiptera: Reduviidae) (Dias et al., 2014), adult insects of T. peregrinus.
Biosecurity measures should be intensified globally in order to hamper the rapid spread of invasive pests and possible damage to plantations. In this sense, we report the expansion of T. peregrinus in Spain and confirm that the population found in Madrid belongs to haplotype A. This is the same maternal lineage found in South America, being one of the probable routes of introduction of this haplotype in Spain.