First record of Diaeretus leucopterus (Haliday) (Hymenoptera, Braconidae, Aphidiinae), the parasitoid of the aphid species, Eulachnus agilis (Kaltenbach) (Hemiptera, Aphididae) in North Africa

A survey of pine-associated aphids and their parasitoids was carried out in the arboretum of Pinus halepensis Miller in the Higher Agronomic Institute Chott Mariem (ISA CM, Tunisia) during 2010–2011 and 2018. The survey revealed the presence of the aphid parasitoid species, Diaeretus leucopterus (Haliday) (Hymenoptera, Braconidae, Aphidiinae). This is the first record of D. leucopterus from Tunisia and the African continent. The parasitoid emerged from the spotted green pine needle aphid, Eulachnus agilis (Kaltenbach) (Hemiptera, Aphididae, Lachninae), on P. halepensis. The generalist hyperparasitoid, Asaphes suspensus (Nees) (Hymenoptera, Pteromalidae), was also found attacking D. leucopterus. The diagnosis of the primary parasitoid, on the basis of relevant illustrations, was provided, and the possible routes of its transportation into North Africa were discussed.


Background
Pinus spp. are the largest genus of the coniferous plants in the northern hemisphere. They include many important species from the economic and ecologic point of views (Gernandt et al. 2005). Three pine tree species are common in Tunisia, including the Aleppo pine, Pinus halepensis Miller; the maritime pine, P. pinaster Aiton; and the stone pine, P. pinea L. The first two species are naturally grown in a wide area of Tunisia. P. halepensis consists of the main forest element of Tunisia that encompasses about 297,000 ha, i.e., 57% of the total forest area of the country (Khouja 1997). P. pinea forests are found along the coasts of Tunisia, in about 27,000 ha, after plantation of dunes between Tabarka and Kelibia.
Although several insect species are associated with the pine forests globally, some of them have been primarily raised as economic pests. Aphids of the genus Eulachnus del Guercio (Hemiptera, Aphididae, Lachninae) have a significant anti-aesthetic impact on the urban areas, where pine species are grown as ornamental and shading plants (Morris 2006). Eulachnus aphids are widely distributed outside their original sources, due to the massive transportation of infested pine stock through international trade (Richardson 1998). Although Eulachnus spp. have a Palaearctic origin, 3 species (Eulachnus agilis (Kaltenbach), E. brevipilosus Börner, and E. rileyi (Williams)) have invaded North and South America (Blackman and Eastop 1994). The majority of these aphids are effectively controlled in their native areas of distribution mainly due to the activity of their natural enemies. However, they become important pests in the absence of their natural enemies in the areas of invasion. Therefore, once established, they cause serious damages to seedling and mature pine trees (Alford 2012 Kavallieratos et al. 2004), there are no data from Tunisia and other North African countries. Till now, only generalist coccinellids have been recorded that associated with Eulachnus spp. in this region (Ben Halima Kamel et al. 2018).
In the present study and through a survey of the pineassociated aphids and their parasitoids carried out in Tunisia, the aphid parasitoid species, Diaeretus leucopterus (Haliday) (Hymenoptera, Braconidae, Aphidiinae), was recorded for the first time. Its diagnosis and the possible routes of its transportation into North Africa were discussed.

Material and methods
The materials were collected from the arboretum of P. halepensis in the Higher Agronomic Institute Chott Mariem (ISA CM, Tunisia) during spring (2010,2011,2018). Pine needles were inspected in situ for aphid colonies (Eulachnus spp.). Once detected, needles, that carried aphids, were carefully cut off and subdivided into plastic boxes with a dimension of 100 × 150 mm. Subsequently, boxes were covered by muslin to allow ventilation of the content. Few adult aphids were separately preserved in 70% ethanol for later identification. The collected materials were maintained for 2-3 weeks under laboratory conditions at 25°C and inspected daily for the emergence of adult parasitoids. The remaining material was kept for 2 additional weeks at the same conditions to rear any secondary parasitoids. The emerged parasitoids were carefully captured, using an aspirator and dropped into 75% ethanol. Female parasitoids were dissected and slide-mounted in the Hoyer medium. The slide specimens were studied under a Nikon® Eclipse E200 microscope (Nikon Corporation, Japan). Photos from the slides were captured by a Canon® EOS 700D camera (Canon Inc., Japan) and used to prepare line drawings in the Adobe® Illustrator CS5. A card-mounted specimen was imaged by using the Canon® EOS 700D, mounted with an adapter on Hund® stereomicroscope (Wetzlar Inc., Germany). A series of 10-15 multifocused captured photographs were subsequently merged into a single in-focus image by using the Zerene Stacker version 1.04 software (Zerene Systems LLC, 2017). The morphological terminology used in this study follows that of Sharkey and Wharton (1997)

Taxonomic account
Coloration: Generally dark, head and mesosoma black, gaster dark brown. Antenna brown, with dark brown scape and pedicel. Legs light brown with yellow patches at tip of segments. Mummified aphids of yellowish brown color were mainly attached to the underside of the needles (Figs. 3a, b). Parasitoids were emerged 7 days post-collection by cutting a circular hole at the posterior part of the mummy above the cauda (Fig. 3c).
The hyperparasitoid Asaphes suspensus (Nees) (Hymenoptera, Pteromalidae) was also emerged from the mummified aphids (2♀ 3♂, same data as for the parasitoid, April 2010 and 2011). This species was already recorded in North Africa in Morocco (Vago 2002). It is a generalist hyperparasitoid of numerous aphid species excluding pine aphids (Gibson and Vikberg 1998). The emergence hole of the adult hyperparasitoid, with irregular margines, is distinctly smaller than the corresponding hole of the primary parasitoid and cutted at the anteriodorsal area of the mummy (Fig. 3d). Another species, Asaphes vulgaris Walker (Hymenoptera, Pteromalidae), was also recorded as a hyperaprasitoid of D. leucopterus via E. rileyi (Suay Cano et al. 1998). Further records regarding the associations of the generalist hyperparasitoids of the genus Dendrocerus Ratzeburg (Hymenoptera, Megaspilidae) with D. leucopterus, both in Eastern (Takada 1973) and Western (Starý 1977) Palaearctics, indicate the presence of diverse food webs rooted on Eulachnus spp. to be explored by subsequent studies. Diaeretus leucopterus was recorded for the first time not only in Tunisia but also in the African continent in association with E. agilis on P. halepensis. Diaeretus leucopterus is a widely distributed species in the Western Palaearctic, including some European countries (Mackauer and Starý 1967;Kavallieratos et al. 2004;Kaliuzhna 2014) and some countries of the Eastern Palaearctic (Starý and Schlinger 1967;Takada 1968;Chen and Shi 2001;Starý et al. 2010) or Oriental (Starý and Ghosh 1983). Several Mediterranean countries, including France, Greece, and Italy, have been recorded as common areas of distribution of D. leucopterus, from where it might be accidentally introduced into the North African area (Tunisia). According to Sghaier and Claustriaux (2015), P. halepensis is of mixed origin since some P. halepensis forests are assumed to originate from the nearby countries, i.e., Africa (Morocco), Southern Europe (Italy), and Western Asia (Israel).
Siphonatrophia cupressi (Swain) (Hemiptera, Aphididae, Aphidinae) has been recently recorded in Tunisia on Cupressus sempervirens L. parasitized by an unknown parasitoid species (Ben Halima Kamel and Mdellel 2017). Since both aphids (E. agilis and S. cupressi) and their host plants (P. halepensis, C. sempervirens) were found in the same area (ISA CM), the association of D. leucopterus with S. cupressi seems expectable. It should be noted that the 2 aphid genera, Eulachnus and Schizolachnus Mordvilko (Hemiptera, Aphididae, Lachninae), were recorded as hosts of D. leucopterus in the Mediterranean area (Kavallieratos et al. 2004). However, further field efforts are necessary to confirm this hypothesis. Few host records of D. leucopterus including Mindarus abietinus Koch (Hemiptera, Aphididae, Mindarinae) (Čapek 1960) and Brachycaudus cardui (L.) (Hemiptera, Aphididae, Aphidinae) (Fulmek 1968  found in the Mediterranean counties (Tremblay 1975;Núñez-Perez and Tizado 1996). It is an uncommon species, while its occurrence in North Africa has not been documented yet. The Nearctic aphids of the genus Essigella del Guercio are biogeographically associated with pine trees in North America (Blackman and Eastop 1994;Théry et al. 2018). Diaeretus essigellae Starý & Zuparko (Hymenoptera, Braconidae, Aphidiinae) was described from Essigella californica (Essig) (Hemiptera, Aphididae, Lachninae) on Pinus spp. in California (Starý and Zuparko 2002). It is closely related to D. leucopterus, but it is immediately separated by the absence of propodeal carinae. However, it is rather unlikely to occur in Tunisia, since pine seedlings are not transported from North America to Tunisia. A comparison of both Diaeretus species, using DNA sequences, can provide further data about the identity of these taxa and their associated host aphids.

Conclusion
Both Eulachnus aphids and their parasitoid, D. leucopterus, seem to be invasive species in North Africa (Tunisia). Considering the economic importance of these pest aphids, further investigation in the neighboring countries is needed to reveal the expansion of the parasitoid and to confirm its possible establishment.