Tachinid (Diptera, Tachinidae) parasitoids of Lobesia botrana (Denis & Schiffermüller, 1775) (Lepidoptera, Tortricidae) and other moths

Abstract The present paper reports data on the biology of eleven species of tachinid flies collected in Italy and Spain on different host plants and emerged from different host larvae. An annotated list of the eleven species emerged from the collected lepidopterans is provided; information about distribution and biology are given as well as the description of their puparia. Two new parasitoid species of the European Grapevine Moth (EGVM) Lobesia botrana were recorded: Clemelis massilia, whose host preferences were unknown so far, and Neoplectops pomonellae. A list of lepidopteran pest species with their associated plants and tachinid parasitoids is then given in order to highlight the relationships among the three components of the biocenosis (plant, herbivore and parasitoid). Eventually, due to the great economic importance of L. botrana in viticulture, a preliminary identification key to the puparia of its tachinid parasitoids is provided.


Introduction
Every year world agricultural yield is reduced by 10-16% both by pre-and post-harvest pests (Bradshaw et al. 2016). Crop losses caused by arthropods appear to be higher in modern industrial agriculture than in traditional agriculture which employs more environmentally friendly and sustainable practices (Culliney 2014, Lucchi andBenelli 2018). Every year, 35 million hectares of forest, especially in temperate and boreal areas, are damaged by outbreaks of harmful insects (FAO 2010, Kenis et al. 2019. With the increase in the volume and speed of international trade, together with climate change, the incidence of these outbreaks is also increasing (FAO 2010, Kenis et al. 2019). Lepidoptera is one of the main groups of plant feeding insects which can be potentially dangerous to both agriculture and forestry.
Biological control is a sustainable and environment-respectful method used for the containment of harmful insects. Among the biological control agents (BCAs), in most cases parasitoid insects are renowned for their effectiveness and specificity. The main orders of insect parasitoids are Hymenoptera and Diptera. Notwithstanding the great knowledge that has been acquired on this topic, many aspects of the parasitoid behaviour and action towards the host are still awaiting clarification. In this context, taxonomy plays a key role for a better understanding of the species to be used as BCAs, and their relative host range. Sometimes rather large host ranges may be an indication of a poorly investigated parasitoid taxon. In other cases, however, they can be due to inaccurate identifications of both the host or the parasitoid, as often occurs with old reports. Presenting data on host-parasitoid relationships can help verify and possibly confirm old records. Moreover, they also increase the amount of data available for future studies focusing on the host range extension and its possible variation under different regional conditions. Among Diptera, Tachinidae is a megadiverse family, representing one of the most diverse lineages of parasitoids (Stireman et al. 2019). Despite being a wellinvestigated family in Europe, some biological aspects, mostly concerning their host-association, are still poorly known (Mückstein et al. 2007). Most of the biological information available so far was recently resumed in the Preliminary Host Catalogue of Palaearctic Tachinidae (Tschorsnig 2017), making comparative studies easier to develop. Among all the tachinid hosts, the European Grapevine Moth (EGVM), Lobesia botrana (Denis & Schiffermüller, 1775), represents a key pest in viticulture (Ioriatti et al. 2011); this moth is present in the grape-growing regions of Europe, Near East, southern Russia, northern and western Africa, and it was accidentally introduced in North and South America (Ioriatti et al. 2012, Cooper et al. 2014. The most common wild host plant of EGVM is the spurge flax, Daphne gnidium L. (Thymelaeaceae), which possibly represents its native host from which EGVM later expanded towards vineyards (Scaramozzino et al. 2017a). Both grapevine and D. gnidium are hosts of other moths, which can be in turn exploited by the same parasitoid community associated with EGVM (Villemant et al. 2012, Scaramozzino et al. 2017b).
In the present paper we report parasitoid-host records for eleven species of tachinids collected in Italy and Spain on EGVM and other lepidopteran hosts feeding on different plant species. Three new host records are here reported: Clemelis massilia Herting, 1977 developing on L. botrana (Denis & Schiffermüller, 1775) living on shoots and inflorescences of D. gnidium in Tuscany, Clausicella suturata Rondani, 1859 on Ephestia unicolorella subsp. woodiella Richards &Thomson, 1932 andeventually Neoplectops pomonellae (Schnabl &Mokrzecki, 1903) on L. botrana. The parasitoidhost issue is addressed on three different levels: first we report parasitoid-host records for eleven species of tachinids collected in Italy and Spain on various lepidopteran hosts; then we refer to the parasitoid-host relationships between plants and different lepidopteran species; lastly, due to the great importance of L. botrana in viticulture, we provide a preliminary identification key to the puparia of its tachinid parasitoids.

Materials and methods
Tachinid flies emerged from caterpillars collected in Piedmont, Tuscany, Apulia and Spain on plants belonging to three different plant families: Quercus ssp. (Q. pubescens and Q. robur) (Fagaceae) in Piedmont, grapevine (Vitis vinifera, Vitaceae) in Piedmont, Tuscany and Apulia, and Daphne gnidium (Thymelaeaceae) in Tuscany and Spain. Puparia were studied by PLS whereas the adult flies, once emerged, were mounted on pins and identified by PC. Moths were mounted on pins and identified by Graziano Bassi, AuL, AnL, and PLS.
Digital images were taken on a Leica Z16 APO stereoscope equipped with a Nikon D5300 digital camera and stacked in a single in-focus image using Helicon Focus 3D (version 3.9.7W) and Zerene Stacker software (version 1.04). All specimens are currently preserved in the collection of the Department of Agriculture, Food and Environment of Pisa University.
Section A -Annotated list of the Tachinidae records. The list follows an alphabetical order. Subfamily, tribe, species name, label information, distribution, biological information and puparium description are reported. Additional information may be found under 'Notes'. Tachinid subfamily, tribe and general distribution are listed accordingly to O'Hara et al. (2019) and Cerretti (2010) for the Italian distribution. Biological and host record information for each species are based on Tschorsnig (2017) and on Cerretti and Tschorsnig (2010). The morphological terminology and characterisation of the tachinid puparia follow Greene (1922), Ziegler (1998) andO'Hara (2005).
Section B -Annotated list of records by host plant and Lepidoptera. The list by host plant and Lepidoptera follows an alphabetical order. The lepidopteran species names refer to Fauna Europaea (de Jong et al. 2014).
Section C -Preliminary key to the puparia of tachinid flies associated with L. botrana. The key is based both on direct observations and on illustrations already available in the literature.
Biology. Parasitoid on a wide range of Lepidoptera and Hymenoptera Symphyta. In Italy it has already been reported on E. (Euproctis) chrysorrhoea (Linnaeus, 1758) (Erebidae) in Emilia-Romagna (Faggioli 1937;Cerretti and Tschorsnig 2010) and Sardinia (Delrio and Luciano 1985). Puparium ( Fig. 1C-E): cylindrical with rounded posterior edges, subshiny, dark red, smooth with circular anterior spinose bands; posterior spiracular plates slightly above level of longitudinal axis and scarcely raised above surface of puparium; each posterior spiracular plate with three linear openings; button round, scarcely defined; anal opening dark, below longitudinal axis at about the same distance of posterior spiracular plates from longitudinal axis.
Notes. Two adults emerged from the same host larva; the larvae pupated within the body of the host larva and the adults emerged from cut-like openings made on the host exoskeleton.

Distribution.
Palaearctic. Italian distribution: north and south Italy, Sardinia. Biology. Parasitoid on a wide range of Lepidoptera and seldom Hymenoptera Symphyta. In Italy it has already been obtained from T. viridana Linnaeus, 1758 (Tortricidae) on Q. robur in Sardinia .
Puparium ( Fig. 3A-D): cylindrical with rounded posterior edges, dull, light brown, surface transversally striated, with circular anterior spinose bands; posterior spiracular plates on longitudinal axis and scarcely raised above surface of puparium; each posterior spiracular plate with four serpentine openings; button round and large; anal opening concolourous, below longitudinal axis, located at some distance from posterior spiracular plates. Distribution. Palaearctic and Oriental. Italian distribution: north and south Italy, Sicily, Sardinia.

A5. Bessa parallela (Meigen, 1824) -First record on T. viridiana in Italy
Distribution. Palaearctic and Oriental. Italian distribution: north and south Italy. Biology. Parasitoid mainly on Lepidoptera, with Coleoptera or Hymenoptera Symphyta as unusual hosts. It has already been recorded on T. viridana in several regions of North, Central and East Europe. This is the first record for this species on T. viridana in Italy. This species is also recorded for L. botrana (Tab. 2), even if the single record in literature (Jordan 1915) is from specimens obtained from lab parasitisation tests (Tschorsnig 2017).

A7. Nemorilla maculosa (Meigen, 1824) -First record on L. botrana in Italy
Biology. Parasitoid of a wide range of lepidopteran families. It has already been obtained from L. botrana in Bulgaria (Trenchev 1980), Iran (Shoukat 2012), Spain (Coscollá 1981) and Ukraine (Telenga 1934). This is the first record for this species on L. botrana in Italy. In Morocco, N. maculosa was found on Cryptoblabes gnidiella (Millière, 1867) (Pyralidae), which often cohabits the same nests built by L. botrana on D. gnidium (Scaramozzino et al. 2017b). The biology and preimaginal stages of N. maculosa have been studied and illustrated in detail by Mellini (1964).
Puparium (Fig. 7E-I): sub-cylindrical with posterior edge slightly depressed dorsally and broadly rounded ventrally, shining, yellow, smooth with not well defined circular anterior spinose bands; posterior spiracular plates clearly above longitudinal axis and slightly raised above surface of puparium; each posterior spiracular plate with three small linear openings and with some scars in between; button round and large, defined; anal opening red, below longitudinal axis and remote from posterior spiracular plates, half the distance from ventral surface.
Notes. According to Tschorsnig (2017), records of Nemorilla floralis (Fallén, 1810) on L. botrana (Telenga 1934, Trenchev 1980, Coscollá 1981) are probably misidentifications for N. maculosa. The puparium of this tachinid was found inside the cocoon of L. botrana, next to the remains of the chrysalis or the mature larva (Fig. 7C). Moreover, during our observations, we found the eggs of a tachinid (Fig 7D) on the pronotum of two mature EGVM larvae. The first larva only had one egg from which emerged a malformed and unidentifiable tachinid fly, though the remains of its puparium were very similar to those of N. maculosa. The second larva bore two tachinid eggs but it unfortunately died before parasitoid emergence. This species was obtained from L. botrana in two different periods of the year: in June, and from the last days of August to the end of September.  Biology. Parasitoid on about 30 hosts belonging to different lepidopteran families. In Italy, it is a renowned L. botrana parasitoid (Scaramozzino et al. 2017a) and it is considered one of the main control agents of L. botrana in the vineyards, where it can significantly contribute in reducing the summer population of the moth (Bagnoli and Lucchi 2006;Thiéry et al. 2006); it has been obtained from L. botrana in vineyards in Piedmont (Colombera et al. 2001), Trentino (Catoni 1914), Veneto (Marchesini and Dalla Montà 1992;1994), Tuscany (Bagnoli and Lucchi 2006), Campania (Silvestri 1912), Calabria (Laccone 2007) and Apulia (Laccone 1978) and from L. botrana nests on D. gnidium in Apulia (Nuzzaci and Triggiani 1982) and Sardinia ). In Spain, it has been reported from L. botrana in vineyards by Coscollá (1981). It also parasitises Eupoecilia ambiguella (Hübner, 1796) (Tortricidae), another important pest of the grapevine. The biology and preimaginal stages of P. nigrina have been studied and illustrated in detail by Mellini (1954), and the life-history was briefly reviewed by Andersen (1988).
Puparium (Fig. 8A-E): sub-cylindrical with both edges slightly depressed dorsally and broadly rounded ventrally, shining, red-brown, smooth with scarce spines towards edges; posterior spiracular plates slightly above level of longitudinal axis, borne on a subconical projection; posterior spiracular plate reduced, without openings; anal opening round and dark, below longitudinal axis and remote from posterior spiracular plates, half distance from ventral surface. It was covered with remains of host larva cuticle.
Notes. Only one specimen of P. nigrina was obtained from EGVM larvae during a 4-year survey on D. gnidium in San Rossore Natural Reserve (Tuscany). In this context, Actia pilipennis resulted instead the most abundant species of Tachinidae parasitising EGVM. Contrariwise, in other researches on the same plant, it was definitely the most common species among the parasitoids of L. botrana (Nuzzaci andTriggiani 1982, Luciano et al. 1988); in Apulia it attacked 30% of the larvae, in Sardinia it was the most common parasitoid on spurge flax while it was completely absent on the vine. In our occasional samplings on D. gnidium in the north of Spain (Girona, Catalonia) during the summer, P. nigrina was the only parasitoid obtained from EGVM in summer. Fig. 9 Label information. Italy, Tuscany: Pisa, Terricciola, vineyard, 08.iii.2006, emerged 20.iii.2006, ex Ephestia unicolorella subsp. woodiella on Vitis vinifera (bark), A. Lucchi leg., 6♂♂ 8♀♀, P. Cerretti det.
Puparium (Fig. 11C-G): suboval with rounded posterior edges, shining, orangeyellow, smooth with incomplete anterior bands of spines; posterior spiracular plates on the longitudinal axis and borne on a cylindrical projection; posterior spiracular plate small, with three small linear openings; button round and small, defined; anal opening concolourous, just below the longitudinal axis.
Notes. In Piedmont, from 1986 to 1988 A. pilipennis was the tachinid most frequently attacking T. viridana larvae on oaks and it was found inside the host cocoons in the rolled leaves. In San Rossore, it resulted the most abundant tachinid parasitoid of L. botrana on D. gnidium, as above mentioned. Normally its puparia are found inside the cocoon of the EGVM, near the remains of the host larva (Fig. 11A, B), and only rarely the fly emerges from the mature larva that has not yet woven its cocoon and pupate between the leaves of its nest.

Species on Quercus spp. [Fagales, Fagaceae]
In Europe, as well as throughout the northern hemisphere, oak is an important component of deciduous forests, representing an extremely species-rich tree. In Britain, Southwood (1961) reports 284 insect species associated with oak, most of them belonging to the order Lepidoptera and Coleoptera (237 species) (Morris 1974). For Western Palaearctic, Soria (1988) lists 453 species of foliage-feeding Lepidoptera, belonging to 37 families. Not all insect species found on oak trees are primarily associated with these plants though, and even fewer are those who can cause considerable damage. Indeed, Klimetzek (1993) reports 136 insect pests associated with oak in Europe. Both the brown tail moth (Euproctis chrysorrhoea) and the green oak leaf-roller (Tortrix viridana) are considered two of the main pests of oak in Europe (Day and Leather 1997).

B3. Cacoecimorpha pronubana (Hübner, [1799]) (Lepidoptera, Tortricidae, Tortricinae)
Eight species of Tachinidae are reported on C. pronubana, two of which have been found in Italy (Cerretti andTschorsnig 2010, Tschorsnig 2017 Larvae of this species can be found inside bunches of grapes and feed on the dried berries. They hibernate as mature larvae in the cocoon, on the woody parts of the vine or on the support poles. So far, no tachinids have been found on this species (Tschorsnig 2017 C. Preliminary key to the puparia of tachinid flies associated with Lobesia botrana The present key includes a strict selection of species, mainly based on the ones directly raised for this study. Puparia of Neoplectops pomonellae are unknown; description of the puparium of Elodia morio is based on Zuska (1963). Posterior spiracular plates with tree linear openings (Fig. 11G). Posteromedian projection subcylindrical (Fig. 11F). Puparium suboval in shape ( Space between the two posterior spiracular plates as long as the diameter of a spiracular plate (Fig. 12C). Spiracular plates with small button (Fig. 12C) Space between the two posterior spiracular plates long less than half the diameter of a spiracular plate (Fig. 4F). Spiracular plates with large button (Fig.  4F)  Posterior end of puparium, in lateral view, almost hemispherical, i.e., posterodorsal and posteroventral portions of puparium (with respect to posterior spiracles) roundly convex (Fig. 5C); spiracular plates arising at about level of midline of puparium in lateral view. Posterior spiracular plate with tree linear openings (Fig. 5E)  Posterior spiracular plates flat, lying on surface of puparium, with tree sinuous openings (Fig. 6F)

Discussion
Both Lepidoptera and Tachinidae play a crucial role in agriculture and forestry, the first as pests and the second as potential BCAs. Therefore, information about parasitoidhost relationships may help in better understanding population dynamics of potential pests in different environments.
In this framework, we provided here eleven parasitoid-host records for tachinids in Italy and Spain. Some are new regional records for Italy, i.e., Compsilura concinnata on Euproctis chrysorrhoea, Carcelia falenaria on Amata sp., and Phryxe cf. nemea on Tortrix viridana, all collected on their hosts in Piedmont for the first time. Pseudoperichaeta nigrolineata, Bessa parallela, and Nemorilla maculosa are recorded for the first time in Italy on their renown hosts, i.e., Cacoecimorpha pronubana, T. viridana, and Lobesia botrana respectively. Clausicella suturata and Neoplectops pomonellae are reported for the first time on Ephestia unicolorella subsp. woodiella and L. botrana, respectively. The record of Clemelis massilia on L. botrana represents the first host record for this species so far.
Three out of these eleven species, Phryxe cf. nemea, Bessa parallela, and Actia pilipennis, have been obtained from T. viridana, one of the major defoliator pests of oaks in Europe, North Africa and Near East (Boghenschütz 1991). Five of these eleven species emerged from L. botrana, i.e., C. massilia, N. maculosa, Phytomyptera nigrina, N. pomonellae, and Actia pilipennis. So far, six species of tachinids have been associated with EGVM (Martinez et al. 2006, Delbac et al. 2015, Tschorsnig 2017, two of which (i.e., P. nigrina and A. pilipennis) have already been recorded on this pest in Italy (Scaramozzino et al. 2017a). Considering the present records as well as the one from Carlos et al. (2019), which confirms the previous observations made by Forti (in Coscollá 1997) and by Hoffman and Michl (2003), the number of tachinids associated with L. botrana rises to nine (Tab. 1). P. nigrina and A. pilipennis have been reared from L. botrana both in vineyards and on Daphne gnidium (Scaramozzino et al. 2017a), while the other three species, C. massilia, N. maculosa and N. pomonellae, have been obtained only from D. gnidium so far. In Spain, P. nigrina has been obtained from L. botrana in the vineyards (Coscollá 1981) and its presence into tortricid nests on D. gnidium in the wild is recorded here for the first time in the country. Among these nine species associated with L. botrana, P. nigrina certainly appears the most common and is also the most cited in the literature (see Tschorsnig 2017). All the other species seem to be occasional parasitoids, which also live at the expenses of other lepidopterans sharing the same host plant (Tab. 2). During our 3-year survey in the Natural Reserve of San Rossore (Tuscany, Italy), the overall parasitisation rate on preimaginal  stages of L. botrana ranged between 12% and 16%, with tachinids accounting for 2-6% of the parasitoid community (Scaramozzino et al., unpublished data). In this context, they play a role as occasional parasitoids of L. botrana and other moths.