Aganaspis daci, a larval-pupal parasitoid of several tephritid species, was unexpectedly recovered in the Campania region (Southern Italy), where it had not been intentionally released. An integrative approach was used to conduct a comprehensive characterization of this parasitoid, confirming its identification through a comparison with specimens obtained from laboratory rearing. While A. daci emerged from puparia of Ceratitis capitata during this study, its original association was recorded with Bactrocera dorsalis. The presence of A. daci in Italy highlights its successful establishment, possibly facilitated by the recent invasive process of its host, B. dorsalis, offering promising prospects for future tephritids control strategies. It is intriguing to note that the mt-haplotypes found in Italy were only partially shared with those observed in specimens originating from a Spanish rearing, suggesting likely distinct origins for at least part of the Italian population.

Bactrocera dorsalis, biological control, Ceratitis capitata, Medfly, oriental fruit fly, Tephritids

The family Tephritidae (Diptera) commonly recognized as fruit flies, includes numerous damaging and invasive pests that pose a serious concern for agricultural production worldwide (Scolari et al. 2021). These fruit flies have the potential to establish into new areas, leading to ecological imbalances and environmental and economic consequences (Szyniszewska et al. 2014). People movements and import-export trade activities are the main factors that allow the transportation of arthropods from one continent to another (Lichtenberg and Olson 2018; Pace et al. 2022).

Among the fruit flies, Ceratitis capitata (Wiedemann, 1824) and Bactrocera dorsalis (Hendel, 1912) (Diptera: Tephritidae), along with other species belonging to the B. dorsalis complex, exhibit particularly high invasive potential. These entities share common biological traits such as high polyphagy, short life cycles, and excellent adaptive capacities (Pieterse et al. 2020).

Ceratitis capitata, commonly known as the Mediterranean fruit fly (Medfly), is native to sub-Saharan Africa. Since its initial discovery in some Southern European countries in the 19^th century, this species has rapidly spread to several countries worldwide, often using European countries as bridgeheads. Additionally, this species is increasingly being detected in areas formerly free of infestation, such as Florida and California, where control and eradication strategies are being implemented (Calla et al. 2014; Szyniszewska et al. 2014).

On the other hand, B. dorsalis, native to Asia, has invaded a significant portion of the African continent (Goergen et al. 2011). It was first intercepted in Italy (Campania Region) in 2018 (Nugnes et al. 2018) and subsequently in 2019 (Gargiulo et al. 2021). However, active infestations of this species on different fruits were recorded in Italy for the first time in 2022, indicating a considerable shift in the scenario, and posing a significant challenge to the agriculture of several European countries (Mertens et al. 2022; Bernardo et al. 2023a; Nugnes et al. 2023). Both entities, B. dorsalis complex and C. capitata exhibit a wide host range, causing damage to 300 plant species including various fruits and vegetables (Gilioli et al. 2022). However, it is important to heed that the accurate host range of the B. dorsalis complex remains a challenge due to possible confusion with related species (Mertens et al. 2022).

The damage caused by these species is mainly due to larval trophic activity, which ultimately leads to fruit collapse and can result in the loss of fruits of high commercial value such as Citrus spp., Malus spp., Diospyros spp., and Prunus spp. (Shelly 2014).

Implementing phytosanitary measures is crucial for controlling these harmful species. Furthermore, as the number of active ingredients and the amounts allowed in cultivation continually decrease, an integrated approach that includes parasitoids has become essential (Jacquet et al. 2022).

Classical biological control, accomplished through the introduction of one or more parasitoids from the native area of the phytophagous pest, often represents the most effective and cost-efficient approach (Moore 2021). However, the effectiveness of natural enemies can vary and be influenced by abiotic conditions, with the species’ thermal requirements determining their success or failure (Adly 2016).

Biological control offers significant advantages, including enhanced security and cost-effectiveness. Numerous successful cases of tephritids management using parasitoids have already been documented, particularly in subtropical and tropical regions (Wharton 1997; Purcell 1998; Ovruski and Aluja 2000).

However, the introduction of exotic parasitoids for biological control purposes is a complex process that involves strict regulations and extensive preliminary studies. Conducting risk assessments can be time-consuming and expensive, resulting in bureaucratic challenges and delays in obtaining authorization for the release of biocontrol agents (Gay 2012; Barratt et al. 2018; Bernardo et al. 2023b).

However, the initial step of this lengthy process involves studying the indigenous parasitoid complex that has rapidly adapted to invasive pests. In addition, the parasitoid complex, both on endemic and invasive species, is constantly evolving, both qualitatively (different species) and quantitatively (varying percentages of relative parasitisation over time and space) (Mirchev et al. 2004). The parasitoid complex associated with tephritids present in Europe (Clausen et al. 1965; Papadopoulos and Katsoyannos 2003; Viggiani et al. 2006; Sasso et al. 2019) has been strongly influenced by the release of numerous parasitoids by Professor Silvestri (Silvestri 1916; Silvestri 1938) and other scientists (Marucci et al. 1952; Bokonon-Ganta et al. 2019; Wang et al. 2021; Coelho et al. 2023).

The aims of this study were manifold. First, we aimed to report the results of a survey conducted on parasitoids developing on C. capitata and/or B. dorsalis in Campanian fields. Second, we aimed to characterize the recorded parasitoid using a comprehensive integrative approach. This included a comparison with specimens obtained from a laboratory rearing in Spain, which, in turn, allowed us to infer the possible origins of the Italian population. Therefore, we assessed the establishment and distribution of this parasitoid in Campania (Italy), with a particular focus on territories affected by the recent invasive process of B. dorsalis. Lastly, the potential implications of the presence of the parasitoid in Italy for future tephritids control strategies are discussed.

Aganaspis daci is a solitary larval-pupal endoparasitoid of tephritids, recognized as an effective biocontrol agent (de Pedro et al. 2021). It was recorded for the first time in Malaysia and Taiwan as a parasitoid of B. dorsalis and subsequently released in Hawaii for the biocontrol of the same pest (Weld 1951; Clausen et al. 1965).

Additional releases of A. daci were carried out in various countries for the biological control of fruit flies such as B. dorsalis, and species belonging to the genus Anastrepha Shiner (Diptera: Tephritidae) (Clausen et al. 1965; Andleeb et al. 2010; Adly 2016; El-Heneidy 2019). Due to successful laboratory assay, the parasitoid has also been released in Egypt for the biocontrol of Bactrocera zonata (Saunders) (Diptera: Tephritidae), the peach fruit fly (Adly 2016; El-Heneidy 2019).

In Europe, A. daci was introduced in France in the 1970s to control C. capitata, but the results were inconclusive (de Pedro et al. 2021). Subsequently, it was recorded in the Greek Island of Chios and Spain, as a parasitoid of medfly, and its presence has not been reported in other European countries (Papadopoulos and Katsoyannos 2003; Verdú et al. 2011).

During the monitoring of B. dorsalis and other fruit flies, a few individuals of A. daci were found in the medfly-infested fruits in Italy. This discovery of A. daci represents the first documented finding of this Figitidae species in the country and highlights the expansion of its distribution range in Europe. The intricate history of its releases and findings, both in Europe and other countries within the Mediterranean region, complicates the reconstruction of its diffusion process, leaving its arrival and establishment unclear. Nevertheless, certain evidence permits to formulate hypotheses. Morphological and molecular analyses confirmed that the Italian and Spanish populations belong to the same species. However, mitochondrial studies suggest that only a single haplotype is shared (Hc) between Italian and Spanish individuals. This indicates a potential difference in the origin of at least a part of the Italian population. However, it is essential to consider that this difference might also be linked to the bottleneck phenomenon, particularly in terms of rearing practices, leading to a decrease in the variety of haplotypes (Prentis et al. 2008). Nevertheless, due to the limited number of Spanish specimens analysed, this result may not be conclusive.

Moreover, consistently with other instances of invasive Hymenoptera, where genetic analysis has indicated that invasive events are frequently attributable to populations comprising a solitary or a few haplotypes, the conducted analyses have revealed the presence of at least three haplotypes in Italy (Nugnes et al. 2015; Nugnes et al. 2016; Sabbatini et al. 2019; Sthal et al. 2019). Nonetheless, it is important to note that these analyses have been conducted on a relatively small number of specimens. Thus, the presence of other haplotypes could not be excluded a priori. Furthermore, the absence of similar sequences in the genetic databases prevented a comparison with other samples from different regions. For this reason, it is not possible to estimate the possible origin of the adult wasp here studied. The absolute absence of genetic sequences belonging to a species found and studied in multiple countries is highly unusual. Therefore, the molecular characterization of A. daci presented here, using a mitochondrial region (COI) and two nuclear regions (ITS2-28S_D1_D2), not only represents the characterization of the entities found in Italian areas but also serves as a starting point for potential identifications and genetic studies by other researchers engaged in studies on this species and its congeneric entities.

The hypotheses regarding the arrival of A. daci in Italy include independent migration from neighbouring countries where the species is already present (Papadopoulos and Katsoyannos 2003; Verdú et al. 2011; Viggiani 2001; Beltrá and Soto 2011; Nugnes et al. 2016), host-tracking, following the invasion of its host fruit fly species (Radeghieri et al. 2002; Gebiola et al. 2014), or a combination of both. It is indeed plausible that A. daci may have arrived in Italy even together with its major host (B. dorsalis) which has been recently and repeatedly intercepted in recent years in Europe (Nugnes et al. 2018; Egartner et al. 2019; Vitiello et al. 2020).

The polyphagy of A. daci suggests that it could have established in Italy by reproducing on C. capitata. However, taking into account the number of Italian tephritids, which were probably not studied in depth because they are not related to agriculture, it cannot be excluded that A. daci may have adapted and reproduced on other hosts. Further investigations are required to determine the exact pathway of introduction and establishment.

The recorded sex ratio (0.56) is consistent with the previously calculated range (0.54–0.61) when the parasitoid was reared on C. capitata, as reported by de Pedro et al. (2017a).

The performance of A. daci as a biocontrol agent has been extensively studied (de Pedro et al. 2016, 2017a; El-Heneidy et al. 2019). Despite its female low fertility and longevity, A. daci can induce pupal mortality, resulting in significant reductions of pest populations. However, its effectiveness depends on environmental conditions, with high temperatures (30–35 °C) and low temperatures (about 15 °C) adversely affecting its development and survival (de Pedro et al. 2016). Conflicting reports exist regarding its field activity, with higher parasitism rates observed in some areas compared to others, and several reports indicating very low parasitisation percentages (Papadopoulos and Katsoyannos 2003; Ali et al. 2016; De Pedro et al. 2017b; El-Heneidy et al. 2019; Moraiti et al. 2020; de Pedro et al. 2021).

The presence of A. daci in Italy from September to December and again in May and July suggests that the parasitoid may be cable of completing more than one generation per year, particularly during seasons with favourable environmental conditions for its development. The observations made in May and July, following the winter period, further confirm its establishment in Italy.

The expansion of C. capitata northward in Italy, driven by climate change, may potentially enable A. daci to shift its distribution to other suitable environments.

The Mediterranean region has proven to be conducive to the establishment of A. daci, as demonstrated by its presence in Spain, Greece, and now Italy (Papadopoulos and Katsoyannos 2003; de Pedro et al. 2016; this work). The discovery of A. daci in Italy not only highlights its natural expansion in Europe, as we have previously discussed, but also its association with C. capitata, which presents a potential additional control strategy not only for this pest but also due to its polyphagy for other fruit fly species.

In the coming years, it will be intriguing to assess the stability of the parasitoid population, its spatial and temporal distribution, and the host range of A. daci, especially in certain Italian locations where another of its host, B. dorsalis, is also found.

It is important to note that European legislation currently imposes restrictions on the introduction of parasitoids for pest control into non-native areas. Despite such regulations, the discovery of A. daci in Italy underscores the ability of natural enemies to traverse the globe and establish in distant territories.

Comparable cases are becoming increasingly frequent (Radeghieri et al. 2002; Beltrá et al. 2011; Gebiola et al. 2014; Gebiola et al. 2015a, 2015b; Nugnes et al. 2016; Stahl et al. 2019).

Therefore, it is essential to reconsider some of these regulations in light of the fact that the arrival of any parasitoid, capable of controlling a phytophagous insect and released in a nearby country, can be prevented. Indeed, the delays caused by the mandatory studies only result in a period in which the alien species can develop undisturbed, leading to the massive use of chemicals that are probably more dangerous for the environment and humans than any parasitoid.

Considering the challenges associated with the importation of B. dorsalis parasitoids, the significance of this discovery cannot be understated. The presence and widespread distribution of its parasitoid in Italy greatly facilitate the implementation of a viable biological control strategy.

The authors are thankful to Francisco Beitia for providing specimens for this study and Fortuna Miele, Laura Figlioli, Giovanna Avventura, Giovanna Ceriello, for their precious technical help.

This study was carried out within the Agritech National Research Center and received funding from the European Union Next-GenerationEU (Piano Nazionale di Ripresa e Resilienza (PNRR) – missione 4 componente 2, investimento 1.4 – d.d. 1032 17/06/2022, cn00000022). This manuscript reflects only the authors’ views and opinions, neither the European Union nor the European Commission can be considered responsible for them. Furthermore, this research was supported by the Campania Region-funded URCoFi project (Unità Regionale Coordinamento Fitosanitario) and by the project ComPLEMEntS (Piante complementari: modulazione dei sistemi entomologici) funded by CNR-DISBA.