Egg parasitoid complex of the pine processionary moth (Thaumetopoea pityocampa) on the Thasos Island, Greece

The egg parasitoid complex of the pine processionary moth (Thaumetopoea pityocampa) was surveyed for the first time on the Thasos Island, Greece. A total of 96 egg batches containing 20391 eggs were collected between 06 and 10 of September, 2017 from Aleppo pines (Pinus halepensis) at four sites (Skidia, Thimonia, Alyki and Panagia). Four primary parasitoids were identified (Ooencyrtus pityocampae, Baryscapus servadeii, Anastatus bifasciatus and Trichogramma sp.), as well as the hyperparasitoid B. transversalis. Among the parasitoids groups, O. pityocampae was the most common, followed by B. servadeii, whilst the number of other species was low. The highest survival rate was reported for three species: O. pityocampae, B. servadeii and A. bifasciatus, while the highest was the mortality in Trichogramma sp. All emerged adults of O. pityocampae and B. servadeii were female specimens and in A. bifasciatus – males. The number of females of B. transversalis was three times higher than the one of males. Ooencyrtus pityocampae and B. servadeii were the most important parasitoids of T. pityocampa, destroying respectively 27.1% and 9.9% of the host eggs.

The aim of this study was to investigate the main characteristics of the complex of egg parasitoids of T. pityocampa from the Thasos Island.

Material and methods
The biological material (96 egg batches of T. pityocampa containing 20391 eggs) was collected on 06-10 September, 2017 from Aleppo pines (P. halepensis) at four sites on the Thasos Island (Table 1).
The Thasos Island is located in the northern Aegean Sea, at a distance of 6.7 km from the continental part of Greece. With an area of 380.1 km², it is the 12 th largest island in this country. The terrain is mountainous and is formed by the Ipsario Mt. with the highest peak at 1203 m a.s.l. Three of the studied sites (Skidia, Thimonia and Alyki) are in the south-east part of the island and Panagia -in the north-east one. The climate is characterised by cool summers and mild, wet winters with average annual temperature 17.2 °C and average summer temperature 23.4 °C in July.
The collected egg batches were transported to the Laboratory of Entomology at the Forest Research Institute in Sofia, Bulgaria. The scales of egg batches were removed and the samples were analysed according to the protocol described in Tsankov et al. (1996). Each egg batch was placed individually in a test tube covered by a cotton stopper and kept under laboratory conditions (20-22 °C). The samples were checked periodically and the emerged parasitoids were removed from the test tubes for identification. In September-October 2018, after the parasitoids completed the emergence, the eggs without openings were dissected and analysed under a stereomicroscope (40×). The parasitoids that had emerged before collection were determined by their meconia and remains according to Schmidt, Kitt (1994), Tanzen, Schmidt (1995), Schmidt et al. (1997a) and Tsankov et al. (1996Tsankov et al. ( , 1998. The statistical analyses of the obtained data were carried out with MS Excel 2013 and Statistica for Windows v. 12.
At three of the studied sites (Skidia, Thimonia and Alyki), the most numerous was the polyphagous O. pityocampae with 63.8-75.5% of all emerged specimens (Fig. 1). The single exception was Panagia (with 6.8%), in which only two egg batches were studied. In the other three samples, the ratio between the two most abundant parasitoids, O. pityocampae and B. servadeii, varied between 2:1 and 3:1. The number of the polyphagous A. bifasciatus was more significant only in Thimonia (1.1%), while at the other sites, it was absent or a single specimen was established. Trichogramma sp. occupied 3.4-9.3% of the parasitoid complex of T. pityocampa with an average of 6.0% for all studied sites. The two most numerous parasitoids of T. pityocampa, O. pityocampae and B. servadeii, were found respectively in 96.9% and 81.3% of the egg batches of the host (Table 2). Trichogramma sp. was recorded from 58.3% of the egg batches of the pine processionary moth. High survival was established in three primary parasitoids. The mortality of O. pityocampae and B. servadeii at different stages of their development in the host eggs was below 5.0%, and no dead individuals of A. bifasciatus were recorded. The mortality of the polyembryonic Trichogramma sp. was significantly higher (76.8%), and of the hyperparasitoid B. transversalis was 11.1%. It should be noted that 792 specimens (8.9%) of the parasitoids had died at an early stage in their development and their identification was impossible.
All emerged individuals of O. pityocampae and B. servadeii were female, while only male specimens were recorded for A. bifasciatus. In the hyperparasitoid B. transversalis, the ratio of females to males was 3:1. A significant part of the adults of O. pityocampae (49.3%) and B. servadeii (29.2%) had emerged before the date of sample collection. On the other hand, all adults of A. bifasciatus and B. transversalis emerged after the collection.
The mortality of T. pityocampa caused by egg parasitoids in the studied areas ranged between 37.3% (Skidia) and 53.5% (Thimonia), with an average of 43.4% for all studied sites (Fig. 2).
Ooencyrtus pityocampae and B. servadeii were the most important parasitoids of T. pityocampa, destroying an average of 27.1% and 9.9% of the eggs, respectively. Unidentified dead parasitoid larvae were found in 3.9% of the host eggs. The rest of the parasitoids occurred in relatively low numbers as follows: A. bifasciatus (0.2%) and Trichogramma sp. (2.4%). They revealed low impact as regulating agents of the pine processionary moth at the egg stage.
The hyperparasitoid B. transversalis parasitized 0.9% of B. servadeii and O. pityocampae on the Thasos Island.

Discussion
In one of the first investigations on egg parasitoids of T. pityocampa in Greece, Kailidis (1962b) reported two species: O. pityocampae and Tetrastichus sp. from the Athens, Thessaloniki and the Attica Regions. In subsequent studies on the Peloponnese Peninsula and in Northern Greece, B. servadeii, A. bifasciatus and Trichogramma sp. were added to the species composition of the primary egg parasitoids of T. pityocampa in the country (Schmidt, 1988(Schmidt, , 1990Bellin et al., 1990). In addition, Bellin et al. (1990) reported Baryscapus sp. near servadeii, which has been described later as B. transversalis (Graham, 1991). Tsankov et al. (1999) found Pediobius sp. as an egg parasitoid of T. pityocampa in the Patra Region (Peloponnese Peninsula). Recently, Mirchev et al. (2010) reported Pediobius bruchicida as a parasitoid of the host in the Drama and the Kastania Regions in Northern Greece.
Outside the continental part of Greece, three egg parasitoids of T. pityocampa were established on the Hydra Island, with a great superiority in the number of O. pityocampae (78.3%), followed by B. servadeii (18.7%) and by B. transversalis (3.0%) (Schmidt et al., 1997a). Bellin et al. (1990) found a significant difference in the ratio between the two most abundant primary parasitoids of the host in Greece: in the Kalogria Region (North-western Peloponnese Peninsula). According the authors, the share of O. pityocampae and B. servadeii was almost equal -52.7% and 47.3%, respectively, while in the Kasandra Region (Northern Greece), the number of O. pityocampae was 3.8-6.2 times lower. In 1997 in Kalogria, the number of B. servadeii was nearly three times higher than the number of O. pityocampae, but in the Athens Region (Southern Greece), O. pityocampae predominated (Mirchev et al., 2010). In this study, O. pityocampae predominated in three of the four studied localities on the Thasos Island. These results differ significantly from data obtained for other regions in Northern Greece: O. pityocampae was more numerous only in the Thessaloniki Region, while at other sites (Drama, Kastania and Asprovalta), a higher share of B. servadeii in the parasitoid complex of the pine processionary moth was reported (Mirchev et al., 2010).
There are several hypotheses that seek to explain the diversity of egg parasitoids of T. pityocampa in different areas. Masutti (1964) has pointed that the representatives of family Eulophidae are more plastic and develop successfully in areas with temperatures above 30 °C, which are not favourable for the development of O. pityocampae. This hypothesis has been supported by the fact that in Italy, the oligophagous B. servadeii is predominant in the warmer regions of the central and southern parts of the country, but it is unexpected that the species has not been found on the Sicily Island and in the pine forests of the Abruzzo National Park at low altitudes (Tiberi, 1990). As concerns polyphagous parasitoids, such as O. pityocampae, it has been suggested that the floristic richness of the habitats is a prerequisite for the high parasitoid numbers, due to the favourable conditions for development of alternative hosts (Mirchev, 2005).
On the Thasos Island, all emerged adults of O. pityocampae and B. servadeii were female specimens. A similar pattern (only emerged females of O. pityocampae and up to 2.2% of emerged males of B. servadeii) has been previously found in other areas of the range of T. pityocampa: Algeria (Tsankov et al., 1995); Morocco (Schmidt et al., 1997b); Albania (Mirchev et al., 1999b); Portugal (Mirchev, Tsankov, 2000); Macedonia (Tsankov et al., 2006); Turkey (Mirchev et al., 2004), Greece (Mirchev et al., 2010). In this study, all emerged adults of A. bifasciatus were male specimens. The development of females of A. bifasciatus in egg batches of the pine processionary moth usually is very rare, but in the Eastern Rhodopes in Bulgaria, 13.8% female specimens of the parasitoid were reared from T. pityocampa .
The ratio of females to males of the hyperparasitoid B. transversalis on the Thasos Island was 3:1. Similar ratio (approximately 2:1) has been reported for the Hydra Island (Schmidt et al., 1997a) and for the Peloponnese Peninsula (Tsankov et al., 1999). In Northern Greece, the share of B. transversalis in the parasitoid complex of T. pityocampa is below 3.0-6.3% (Bellin et al., 1990;Mirchev et al., 2010). In this study, the relative share of B. transversalis was very low: between 0 and 1.6%, with an average of 0.9%. The high values of egg parasitism of T. pityocampa at the studied sites on the Thasos Island (37.7-53.5%) is probably due to the low suppressive impact of the hyperparasitoid on O. pityocampae and B. servadeii.
In conclusion, it should be noted that this is the first study of egg parasitoids of T. pityocampa on the Thassos Island. It extends our knowledge about the biology of the species and their effects on the host.