Replacement of Megastigmus pistaciae Walker (Hymenoptera: Megastigmidae) by Eurytoma plotnikovi Nikol’skaya (Hymenoptera: Eurytomidae) in pistachio orchards in southern Italy

Investigating the effect of non-native herbivore species in the new areas is vital for understanding their effects on native crops and the interactions that the newly arrived species have with any natural native herbivores and predatory species. The pistachio-seed wasp Eurytoma plotnikovi Nikol’skaya (Hymenoptera: Eurytomidae) is an invasive species on pistachio groves in Sicily, southern Italy; thus, the current study investigated its effects on pistachio crops and the native pistachio-seed wasp Megastigmus pistaciae Walker (Hymenoptera: Megastigmidae). The results showed that fruits in Pistacia vera pistachio groves were infested only with E. plotnikovi, which had been able to replace M. pistaciae after only a decade. Female E. plotnikovi emerge between May and June, but the males are rarely seen. No natural enemies were detected for E. plotnikovi in the study sites, highlighting an important phytosanitary issue for pistachio nut production in Sicily.


Introduction
The introduction of non-native species into the new areas far from their natural distribution requires research to determine any direct and indirect impacts they might have in their new environments (McNeely, 2001).Such introductions involve numerous different taxa that perhaps affect most significantly the agricultural industry (Sileshi et al., 2019).Insects include common invasive species, aided in particular by human interventions, such as through international trade or accidental introduction (Seebens, 2019;Venette & Hutchison, 2021).Invasive non-native species often have significant effects on native biodiversity species and ecosystems, posing serious problems for the conservation of populations (Walker & Steffen, 1997).Given the potential for non-native species to negatively influence native populations, communities, and ecosystems, it is vital to determine their occurrence and possible adverse impacts as soon as possible, particularly regarding native species with which they occupy the same ecological niche.The control or eradication of a non-native species is a priority if it negatively influences its host plant, particularly if crop plants are involved (Bonsignore et al., 2021).
Various species and subspecies of the pistachio tree genus Pistacia are grown in southwest Asia and in the Mediterranean basin.The seeds, gums, and galls produced by plants of this genus have been used in southwest Asia since the Epipaleolithic and Neolithic, as evidenced by archeological finds of Pistacia fruits, Vol:.( 1234567890) seeds, and charcoal remains (Rousou et al., 2021).Among the domesticated species, Pistacia vera L., is cultivated on a large scale in different areas in southwest Asia, the Mediterranean basin, and the USA (FAOStat, 2019).Over the past few decades, there has been a change in the framework of pests that can affect the seed production of P. vera, and several species of seed-feeders have been reported, including Amyelois transitella (Walker, 1863) (Lepidoptera: Pyralidae) and two pistachio-seed wasps, namely, Megastigmus pistaciae Walker (Hymenoptera: Megastigmidae) and the invasive species Eurytoma plotnikovi Nikol'skaya (Hymenoptera: Eurytomidae) (Jarraya & Helali, 1978).
Megastigmus pistaciae has been known for years throughout pistachio crops in Italy (De Stefani, 1917, Monastero, 1958-59;Nieves-Aldrey et al., 2008) and in all pistachio-producing countries (Jarraya & Bernard, 1971), including new production areas, such as California (Rice & Michailides, 1988).It has two generations per year with overwintering larvae produced from eggs of both generations (Jarraya & Bernard, 1971;Rice & Michailides, 1988).The second generation appears after shell hardening of pistachio seeds and thus, it is thought that the second generation is unable to predate on pistachio seeds (Taghizadeh, 1953).
Eurytoma plotnikovi expands the overall framework of seed-feeder species harmful to pistachio production; however, its occurrence in the various Mediterranean countries where pistachio production occurs is not always known.It is thought that, in the western Mediterranean (e.g., Jordan), E. plotnikovi was present since the early 1960s (Doğanlar et al., 2009) and was subsequently reported in Tunisia (Jarraya & Helali, 1978), Turkey (Uygun, 1994;Doğanlar et al., 2009), Iran (Basirat & Seyedoleslami, 2000), and Israel (Izhaki, 1998).The species is also reported in the Middle East and in China on Pistacia chinensis (Tian et al., 1994;Li et al., 2008;Tang et al., 2012).The occurrence of this seed wasp in Europe was first recorded from Greece and Italy only in 2009 (Mourikis et al., 1998;Longo & Suma, 2011), and was also recently reported from France (Rousse & Reynaud, 2022).From its initial occurrence in southwest Sicily, E. plotnikovi has spread to all areas of pistachio cultivation on the island, with significant detrimental effects on pistachio harvests.As a seed-feeder, E. plotnikovi is able to completely empty the seed cases, resulting in production losses as high as > 80% of the entire production (Mourikis et al., 1998).Initially, it was unclear which wasp species was causing the damage to the pistachio fruits, M. pistaciae or E. plotnikovi.On hatching, eurytomid larvae feed on the developing endosperm of P. vera seeds.The wasp has one generation per year, and the final instar larvae over winter inside the seed case (Mohammadzadeh et al., 2017).The present study evaluated the occurrence of M. pistaciae in Sicily, after just over a decade from the first finding of E. plotnikovi by comparing infestation levels of the two species in different pistachio orchards in the area of the first original report of E. plotnikovi in Sicily.Natural enemies adapted to E. plotnikovi and their effectiveness to control this pest were also investigated.

Observational orchards
Pistachio seeds were collected from trees in conventionally farmed orchards distributed in different areas (Table 1) within the geographical region where E. plotnikovi was first found by Longo and Suma (2011).The pistachio orchards occur within a Protected Designation of Origin (PDO) of 'Pistachio of Raffadali' that lies within a Mediterranean xeric-type climatic area.Pistachio groves (cv Napoletana) are obtained from the grafting of P. vera on P. terebinthus on regosol soils based on gypsum, chalky clays and calcareous substrates.The orchards are not irrigated and ranged in altitude from 250 to 550 m above sea level.The average temperature in the study area was ~ 18 °C with an average rainfall of 500 mm per year, mainly during the autumn, with a dry summer; the area experiences torrential precipitation events over a period of ~ 5 months (based on data from 1971 to 2000) (Arnone et al., 2013).

Fruit sampling and infestation by seed-feeders
Pistachio seeds that showed evidence of insect attack or other diseases affecting the seeds were collected.In September, mummified and empty pistachio seeds containing fully grown larvae of seed-feeding insects were collected from ~ 20 sites in each study location (Table 1).To separate the fruits with the aforementioned symptoms, in agreement with the farmers, all harvested seeds were added to drinking water and those that floated were removed; alternatively, the seeds were exposed to the action of a mechanical fan for < 60 s and any that were blown by the fan were also removed.
Then, in each study area, 400 seeds were randomly selected from these collected seeds (N = 2000 in total).Seeds were placed individually in polystyrene containers with a fabric lid to allow gas exchanges.The containers were kept in the dark at room temperature and the position of the containers was changed weekly.
Observations on the seeds and separation of any insects that emerged were performed weekly starting from May until August.All emerged insects were observed under a stereomicroscope, separated by site and date, and then preserved in absolute alcohol for storage.

Identification of emerging insects and data analysis
Each insect that emerged from a seed was observed under a stereomicroscope Olympus SZX9 at magnifications from 20 × to 60. Species were identified using a key for the Eurytomidae (Nikol'skaya, 1935;Zerova, 2017).The total number of pistaciae seeds collected and the percentage parasitization of seeds (i.e., the number of seeds from which a phytophagous or its parasitoid emerged) at different sites were used for analysis.SPSS v.23 (IBM, 2015) was used for data analyses and SigmaPlot 13.0 (SigmaPlot, 2018) was used to produce graphs.The images were taken with a Dino-Lite mod AM73915MZTL acquisition and processing system.

Results and discussion
The results highlighted the almost total presence of E. plotnikovi in all study sites, with clear differences between sites, accounting for 5.25% of damaged seeds in area 5 and 13.25% in area 1.The appearance of adult E. plotnikovi occurred during May and June (Fig. 1).Amyelois transitella (Walker, 1863) was present in low numbers only in one location (with only two specimens obtained).The study confirmed the scarcity of male E. plotnikovi (Fig. 2), which were mainly black except for their brownish yellow legs.
The males had antenna with seven-segmented funicles and long pubescence (Fig. 3).In addition, their forewings had marginal veins that were longer than the stigmal vein.
Invasion by E. plotnikovi has resulted in the net reduction of M. pistaciae in the study area, as well as in other Sicilian pistachio cultivation areas where the presence of M. pistaciae is sporadic (personal observation).Despite the presence of many seedfeeding species belonging to the genus Megastigmus Dalman, 1820, which are able to feed on conifer seeds and as well as Anacardiaceae, Malvaceae, and Rhamnaceae (Roques et al., 1999(Roques et al., , 2016;;Roques & Skrzypczyńska, 2003), these species have not yet replaced native seed-feeding species.Therefore, E. plotnikovi is thought to compete more successfully with M. pistaciae in their invaded environment, probably because the native species is also exposed to natural enemies.Similar replacement has also been reported in Tunisia (Jarraya & Bernard, 1971;Braham, 2005;Braham et al., 2010) but needs to be verified in other areas where E. plotnikovi is now established.Diapause in Eurytoma plotnikovi depends on both temperature and photoperiod (Tzanakakis et al., 1992) and this species survives the winter either inside the fruits that remain on the trees after harvest or in those that have fallen on the ground.The final-instar larvae of E. plotnikovi are able to tolerate the low temperatures, experienced mainly during December-February (Mohammadzadeh et al., 2017).In mid-spring, the larvae, which cause significant damage to the growing seeds, pupate inside the nut and adults emerge through a round hole that they bore in the shell (Fig. 4).The seasonal flight period reported in Tunisia starts at the end of April and continues into June (Braham, 2016).Doğanlar and Doğanlar (2010) reported that the parasitoid Gugolzia karadagae (Hymenoptera: Pteromalidae) is able to parasitize up to 17.5% of E. plotnikovi (Doğanlar & Doğanlar, 2010).However, this parasitoid was not found in other areas investigated by the same authors.Therefore, the absence of information on the interactions between E. plotnikovi and its natural enemies is a significant gap in our ability to develop biological control approaches for this pest.
The control of E. plotnikovi and others Pistacia pests in Sicily is generally based on the application of insecticide sprayings during late spring and July.
Given the limited number of insecticides that can be used against this species in Europe (deltamethrin, lambda-cyhalothrin, etofenprox and those derived from natural products, such as spinosoids), and with authorizations that will be modified or revoked, further work is required to understand the life cycle and  natural enemies of E. plotnikovi in more depth.One approach to reducing the presence of phytophagous insects is to bury any dropped fruit.However, this is not always possible in the study area because of the scattered presence of uncultivated pistachio trees, which supports the constant presence of E. plotnikovi in this region.In Sicily, growers use phytoiatric strategies in the most important areas of production, accentuating the alternation of production of pistachio groves through the removal of flowers during the  unloading years of production (Bonsignore, personal communication).Further investigations on other seeds of Pistacia species, such as Pistacia lentiscus and other Anacardiaceae are necessary to verify the presence of M. pistaciae and its possible interaction with E. plotnikovi.