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The Asian hornet (Vespa velutina) is a species included in the Spanish Catalogue of Invasive Exotic Species according to Royal Decree 630/2013, for which there is a management, control and possible eradication strategy. It was first detected in France in 2004 and quickly spread to nearby countries such as Spain. This wasp preys on insects, mainly on bees. The University of the Balearic Islands highlights that its introduction causes significant impacts on biodiversity, affecting pollination in natural ecosystems and causing the decrease of wild insects. It also generates problems in the economic field (mainly on beekeeping and crop pollination) and in the health (due to possible bites).

The route of introduction into Europe from Asia is not clear and neither is its arrival in Portugal, Galicia or the Balearic Islands. According to the entity, to prevent the introduction of this invasive species it is essential to know how and from where it has arrived in each region. In this project, a study has been carried out on their genetic diversity to understand the routes of entry and dispersal, and to propose specific management measures.

The general objective of the project has been to evaluate the entry and dispersal routes of the Asian hornet in the Iberian Peninsula and the Balearic Islands for the establishment of specific management mechanisms for the species.

The specific objectives were as follows:

• To collect samples of Asian hornets from the Iberian Peninsula and the Balearic Islands.
• Extraction of DNA from the samples.
• Quality control of DNA extractions.
• PCR amplification of three key regions of the mitochondrial genome.
• Sequencing of mitochondrial molecular markers.
• Amplification of 22 microsatellite regions of the nuclear genome (STR).
• STR fragment sequencing .
• Bioinformatic editing of DNA sequences.
• Data analysis STR.
• Genetic characterization of individuals from the Iberian Peninsula and the Balearic Islands.
• To reveal the origin of individuals from the Iberian Peninsula and the Balearic Islands and the number of colonization events.
• To evaluate the genetic flow of the species in the Balearic Islands.
• Prepare a document with the information obtained so that it can be included in the Strategy for the Management, Control and Possible Eradication of the Asian Hornet.
• Disseminate and communicate the results and conclusions of the project.

• A compilation of Vespa velutina individuals already collected in the Balearic Islands between 2015 and 2018, obtaining a total of 274 adults.
• Compilation of 61 adult individuals collected in different parts of the Iberian Peninsula (specifically Catalonia, the Basque Country, Asturias, Galicia and Extremadura) and sent to the Zoology and Genetics laboratories of the University of the Balearic Islands.
• Collection of the 335 samples collected in total and storage for processing and molecular analysis.
• Extraction and purification of genomic DNA from the three right legs of the 335 individuals.
• Quantification by fluorimetry of the DNA present in the extractions and analysis of the integrity of the DNA by electrophoresis.
• Amplification of the regions corresponding to the genes cytochrome oxidase I (cox1), cytochrome b (cytB) and ribosomal subunit 16S.
• Bidirectional sequencing using the Sanger technique of PCR products related to the cox1 gene.
• Fine-tuning and amplification by multiplex PCRs of a total of 22 loci of STR repeats with demonstrated resolving capacity in Asian hornet.
• Analysis of the amplified STRs fragments in the ABI Prism 3130 DNA genetic analyzer.
• Analysis of the electropherograms obtained and generation of contigues with the CodonCode Aligner program.
• Genotyping of hornets for the 22 loci studied by analysis in GeneMapper software.
• Bioinformatic analysis of DNA sequences to generate phylogenetic trees and haplotypic networks.
• Analysis of genotypic diversity, comparing the microsatellite data generated throughout the project for the 335 individuals, with an existing dataset containing genotypes of 417 individuals collected in invaded Spanish regions and in native distribution areas of Vespa velutina in Southeast Asia. From these data, different results were obtained on estimated population parameters, such as allele frequencies, observed number of alleles, allelic richness, genetic structure of Asian hornet populations, etc.
• Implementation of the approximate Bayesian calculation method to estimate the genetic origin of invasions and the number of independent colonization events.
• Detailed analysis of the genetic profiles of the Balearic samples of all the nests located and the queens trapped since 2015 to establish the patterns of gene flow and dispersal of the species in this time interval.
• Preparation of a final report of the project in which all the work carried out during the project is explained and detailed and management measures for the invasive species are proposed.
• Dissemination and communication of the project: participation in a regional conference and in 3 international congresses in which the results of the project were presented, in 4 meetings with researchers, managers and interested public where a presentation of the results was also made; making publications on social networks, preparing a scientific article, writing a final report with the results achieved in the project, etc.

The STOP Vespa velutina project has aimed to evaluate the entry and dispersal routes of this species in the Iberian Peninsula and the Balearic Islands, in order to establish specific management mechanisms.

It is an extensive project with an important genomic research work in the laboratory. In this sense, at the beginning of the project, samples of individuals already collected in the Balearic Islands in previous years were collected and prepared for subsequent analysis. A total of 274 adults of Vespa velutina were collected from the invasion of Mallorca, and 61 from the Iberian Peninsula, resulting in a total of 335 samples.

Through mitochontrial and nuclear DNA analysis, it has been possible to find out that there were two independent introductions in the Balearic Islands: the first in 2015 from Italy and the second in 2021 from the Peninsula, mainly from Catalonia. In addition, mitochondrial and nuclear analyses suggest that Vespa velutina populations in both mainland Spain and the Balearic archipelago could have derived from the southward expansion of the population of the species initially established in France.

Likewise, the results indicated that the most likely scenario of invasion in Europe by the Asian hornet in both mainland Spain and the Balearic archipelago could have derived from the expansion of the population initially established in France towards the south (towards Spain and Italy), rather than from multiple independent introductions from the native range. According to the entity, once it entered Spain through the west of the Pyrenees, the invasive populations dispersed throughout the north of the Iberian Peninsula, reaching the autonomous communities of Catalonia and Galicia, finally reaching Portugal. In addition, it is concluded that the Mediterranean islands could not be colonized naturally, and that it could only reach the Mediterranean islands by accidental introduction by humans.

The laboratory work has been complemented with the dissemination of the problem of the species, through different media, both regional and national, with different target audiences, from the scientific community to society in general. Finally, a final report of the project has been prepared, which explains and details all the work carried out during it and proposes measures to add to the management, control and possible eradication strategy of the species.