Natural dispersion

Sampling and field work were conducted within the framework of three scientific projects focused on ACGW in Spain. The main project, developed at the level of the entire state, had the twofold aim of studying the recruitment of native parasitoids and assessing the environmental risks associated with the introduction of T. sinensis . At the same time, similar projects were conducted in the autonomous communities of Andalusia and Galicia.

The chestnut forests of the northern IP form a "continuum" from SW France along the Cantabrian Coast to Galicia. To study the natural dispersal of T. sinensis from France, we selected two sampling sites in Catalonia, six in the Basque Country and two in Navarra known for their heavy ACGW infestation, at the east and west corners, respectively, of the border with France, where the Pyrenees are not an effective barrier and both form biological corridors (Table 1, Fig. 1). These areas are isolated and very distant from the Spanish territories where the T. sinensis 2015-2017 releases were performed. An additional locality was also sampled in the community of Cantabria, close to the Basque Country, but further away (approximately 200 km) from the French border and at about the same distance from Asturias, where controlled releases were carried out in 2017 (Table 1, Fig. 1).

Establishment after authorized controlled releases

To study T. sinensis establishment after controlled releases by the concerned authorities, the field sam­plings were done mainly in Galicia (NW of the IP) and Andalusia, the Spanish regions were the first experimental releases of T. sinensis were authorized and performed. In Andalusia, 8 sites were repeatedly sampled from 2016 to 2018 while in Galicia we sampled 35 sites in 2018. In addition, 2 sites of the Autonomous Community of Asturias were also sampled for this purpose (Table 1).

An experimental release of T. sinensis was perfor­med in 2017 on infested chestnuts of the Royal Botani­cal Garden Alfonso XIII of the Universidad Complutense de Madrid (Madrid, Central Spain). Introduced T. sinensis were provided by "Agrobio enterprise" that imported the specimens from Italy, the same commercial source that supplied the individuals released in Andalusia. On 16 ^th May, 120 T. sinensis (48 males and 72 females), females mated about 10 days old, were released on four branches infested with ACGW galls isolated with gauze sleeves. In February, the isolated galls were collected, moved to the laboratory and kept under indoor conditions until the emergence of T. sinensis.

With the aim of rearing T. sinensis (as well as native parasitoids with a similar life-cycle), whose emergence occurs from dry galls after winter, ACGW galls were preferentially collected during the winter and then transferred to indoor laboratory conditions until emer­gence of the insects. This usually occurred one or two months before the natural outdoor emergence because of the forced conditions of the laboratory. Collected galls were located into cardboard emergence boxes equipped with light extractors and kept in normal indoor laboratory conditions.

For an accurate estimation of the real emergence date of T. sinensis in the field, an additional experiment was performed in Juanar (Málaga, Andalusia). Three infested chestnuts were selected, and the galled branches bagged. A total of 30 bags were placed (10 per tree), encaging an average of 8, 6 and 7 galls per bag. The emergence of T. sinensis was checked weekly.

A careful morphological examination of the rea­red specimens attributed to T. sinensis is essential to prevent any possible misidentification with other native torymid parasitoid species. Confusion is particularly possible with three species that are morphologically quite similar and taxonomically clo­sely related: Torymus notatus (Walker, 1833), Tory­mus cyaneus Walker, 1847 and Torymus affinis (Fons­colombe, 1832), and particular attention should be taken with males, which are less easily distingui­shed. For the identifications, we used the unpublished keys kindly provided by R. R. Askew and C. Thuroczy, as well as additional diagnostic morphological characters (Nieves-Aldrey, unpublished data).

All the galls collected during the study together with their collection dates, the number of T. sinensis individuals recovered per locality, their emergence dates, and parasitism percentages (TsPR) appear in Table 1. To estimate parasitism rate of ACGW by T. sinensis we used the following equation

Navarra, Basque Country and Cantabria (northern IP)

From the samples collected in Navarra and the Basque Country, we recovered 147 females and 73 males of T. sinensis . The species was present in one of the sampled sites from Navarra (Bera de Bidasoa) and in three of the sampling sites in Guipúzcoa Province (Basque Country): Hondarribia, Jaizkíbel and Lezo. Compared with the relatively low figures of parasitism to date, from the Spanish sites where were T. sinensis was artificially released, the parasitism percentages in the Basque Country and Navarra were variable but were generally moderate to high (from 0.99% to 8.29%), the relatively high rates at Lezo (4.88%) and especially at Jaizkíbel (nearly 8.5%) being remarkable. These data unequivocally show the successful establishment of T. sinensis in these two autonomous communities, likely as a result of its natural dispersal from France. All the localities with T. sinensis are very close to the border with France, Lezo being the farthest away (approximately 14 km). In contrast, in the sam­ples collected in three sites in Vizcaya Province (Bas­que Country) (Orozco, Berango and Urdúliz), which are substantially farther from France (approximately 130 km), T. sinensis was not present. In addition, samples of ACGW galls collected in S. Roque de Riomiera, in the neighbouring Autonomous Community of Cantabria (225 km from France), did not yield any T. sinensis individuals.

Catalonia (northeast IP)

Twelve T. sinensis females were reared from ACGW galls collected during the summer in two sites, Sant Mar­çal and S. Hilari Sacalm, in the Natural Park of Mont­seny (Barcelona and Girona, Catalonia). The calculated parasitism rate was 0.96% at the first site and 0.19% at the second (Table 1). The specimens successfully emerged in the laboratory from fresh galls collected eight months before (see Table 1), despite the galls being collected in summer and not subsequently enduring a 'cold' period for larval diapause before pupation.

Andalusia (southern IP)

A total of 275 individuals of T. sinensis (154 females and 121 males) were recovered from 12,218 galls collected in eight sampling sites of Málaga Province, where experimental releases of T. sinensis were made in 2015-2017 (Table 1; Fig. 1). The settlement of T. sinensis was especially successful in Juanar, where 230 individuals were recovered, with an estimated mean parasitism rate of 0.51% (range 0.10-0.86, n = 5), despite the fact that experimental releases were made only in 2015 at this site, with 720 individuals (480 females and 240 males) being released. In addition, 34 T. sinensis individuals were recovered from another two sites, Igualeja and Júzcar, in the Valle del Genal, while we also recovered 3 specimens from Puerto de Ojén, which is not too close to Juanar, both being in the Sierra Blanca, and 8 from Yunquera in the Sierra de las Nieves.

To have available accurate data on the phenology of T. sinensis in the areas where it has been introduced is important to determine the best release date for a successful biological control programme with this species. Accordingly, in the experiment performed in 2018 at the site of Juanar, the emergence of T. sinensis under field conditions began in mid-March and ended in mid-April, with an emergence peak in late March (Fig. 2). Therefore, this date may be the most appropriate for releasing T. sinensis in Andalusia region.

Galicia (northweast IP)

Given the extent and economic importance of the chestnut in Galicia, authorized controlled releases of T. sinensis have been important in this region. Up to 220,395 individuals were released in three campaigns (2015-2017) according to Pérez-Otero et al. (2017) , and even higher numbers were released in the 2018 campaign (704,270 individuals from data of the Xunta de Galicia). However, only 12 individuals have been recovered according to published data, all from release sites in Ourense Province ( Pérez-Otero et al. , 2017 ). Our own data even showed null settlement values at different localities. Indeed, from 4,871 galls collected in the winter of 2017 and early spring of 2018 from 33 sites in Galicia, only one male T. sinensis was recovered at one site (Riós, Ourense) where controlled release began in 2017. In addition, the species was not recovered from galls collected in winter (2017) from several sites of the Ribeira Sacra region (Central Galicia).

Madrid (central IP)

The experimental release of 120 individual T. sinensis on ACGW galls in Madrid returned 25 emer­ged T. sinensis individuals (13 females and 12 males ). The success of the experimental infection was high, with a resulting parasitism rate of 3.69%.

In this work, we report the presence of T. sinensis in northern Spain in three autonomous communities at the eastern and western ends of the border with France: the Basque Country, Navarra and Catalonia. Authorized releases of this biological control agent have been made in recent years in Galicia, Asturias and Andalusia, and thus in geographically distant regions from those mentioned above, but no releases have been made to date in communities close to the border with France. Discounting the possibility that the presence of T. sinensis in these northern autonomous communities is a result of many illegal or uncontrolled releases, the most likely explanation is that T. sinensis has naturally dispersed to these territories from France across the southern border with Spain. Published data of T. sinensis releases in France ( Borowiec et al. , 2014 , 2018 ) show that the closest release sites to Spain are Gironde, Lot, and Hautes-Pyrénées, 172-237 km away from the northern Spanish sampling sites. Taking into account that T. sinensis was released in France in 2013 and that the estimated dispersal capacity of this parasitoid is approximately 70 km per year ( Colombari & Battisti, 2016 ), we suggest that T. sinensis may have reached the ACGW-infested Spanish areas in 2016. Fast natural dispersal of T. sinensis was observed also in Hungary ( Melika et al. , 2017 ).

The hypothesis of an early arrival of T. sinensis in northern Spain by natural dispersal from France is reinforced by the relatively high parasitism rates shown in Lezo and Jaizkíbel (4.8% and 8.2%, respectively). It is largely accepted that T. sinensis needs 5-7 years after the first release and or settlement to reach such high rates of parasitism ( Quacchia et al. , 2008 ; Borowiec et al. , 2014 ; Matošević et al. , 2014 , 2017 ; Ferracini et al. , 2018 ). However, despite this early arrival in the Basque Country and Navarra, our data indicate that the progression into Spain throughout the Cantabrian coast was slow, given the apparent ab­sen­­ce of T. sinensis in the sampled sites of Vizcaya Province and the neighbouring community of Cantabria. The spread of T. sinensis from France towards Spain takes advantage of the existence of two broad ecological corridors connecting both sides of the eastern and western Pyrenees. As a comparison between the two zones, the dispersal of T. sinensis across the western corridor, from France across the Basque Country and Navarra, would be easier than along the eastern corridor connecting France and Catalonia, since the first route has a lower altitude and a milder climate.

With regard to Catalonia, our data confirm the recent publication of the presence of T. sinensis in this autonomous region ( Jara-Chiquito & Pujade-Villar, 2018 ). These authors reported a total of 45 individuals (15 males and 30 females) from three localities in Girona Province, close to the sites sampled by us. However, they do not provide data on the parasitism levels.

The release programmes with T. sinensis carried out from 2015 to 2017 in two large areas of Spain, the whole Galicia region and areas of the Andalusian Province of Málaga yielded different results about its settlement. Whereas in Andalusia, the first successful results came after three years of experimental releases (275 T. sinensis were recovered out of 41,420 released, present results), in Galicia (where releases were even more numerous), only 12 out of 220,395 released individuals were recovered in post-release controls ( Pérez-Otero et al. , 2017 ), and only one in our study. Up to now, all available data have shown a low rate of T. sinensis implantation in this autonomous community. It is expected however, given the massive releases of T. sinensis made by the Xunta of Galicia in 2018 that the situation could significantly change after update the recovery data of 2019. We had not still access to the unpublished data of recoveries from the Xunta of Galicia, but our own data have started to show emergences of T. sinensis from galls collected in additional samplings in 2018-2019 (3 females and 2 males recovered to date (February 2019) from galls collected at the site of Merouzo pequeno (Ourense)).

The settlement seems to be particularly successful in Juanar, the first site in Málaga Province where T. sinensis was experimentally released in 2015. Despite the lack of further releases in the area since then, more than 200 individuals were recovered from test-release controls in 2018, with an estimated parasitism rate of 0.51%, thus suggesting successful establishment. It is important emphasize however, that the release area in Galicia is much wider than the release area in Andalusia, and the relative sampling effort was much lower in the first one, which may be one of the reasons of the low establishment success in that region.

It is interesting to compare the parasitism rate recorded from the different sampling sites. Values were null or low (≈1%) at the release points (Table 1), while they were high at sites in the Basque Country (8.2%), i.e., where T. sinensis populations occur because of natural spread from France. The reasons behind this difference is difficult to explain using collected data. It seems not probable that settlement success depends on the time from the releases, since these were done starting back in 2015, possibly even before the arrival of the parasitoid in the northern regions by natural dispersal from France. On the other hand, we cannot exclude an effect of some unknown environmental and climatic conditions (which are extremely variable across the IP, and particularly between the northern and southern coastal areas) on the settlement success ( Gil-Tapetado et al. , 2018 ). Additional studies are necessary to understand whether the release of T. sinensis is more likely to fail under certain conditions and thus possibly not be useful against ACGW.

The data from the experimental release in Madrid showed successful establishment of the insect in the field, on ACGW galls, which is a promising starting point towards future controlled releases in this and nearby regions where C. sativa is present but infection with ACGW have been not detected yet.

We predict that T. sinensis will continue its spread over the northern part of the IP, following C. sativa forests infested by ACGW, possibly reaching in a few years many more areas than where it actually occurs today.

Established populations of T. sinensis may exert a positive buffer against D. kuriphilus -driven chestnut infestation in Spain, similar to what is observed in other invaded European countries.