Parasitoids of the lime leaf miner Phyllonorycter issikii ( Lepidoptera : Gracillariidae ) recorded throughout the area it recently colonized

Phyllonorycter issikii (Lepidoptera: Gracillariidae) has recently spread over a considerable part of the Eurasian continent. So far, in Europe, unlike in russia, it has not significantly affected its food plant (Tilia cordata), but its effect may increase over time. The regulating potential of associated parasitoid natural enemies is still low, which probably allows further leaf miner range expansion and population growth. This species is potentially capable of inflicting considerable damage on its host. Therefore, it is important to study its natural enemies, especially their oviposition behaviour and biology. In this paper, we summarize the results of rearing experiments throughout the area it has recently occupied, including our own study in Hungary. Here we list 46 species of parasitoids recorded from P. issikii. None of these species are likely to be able regulate the abundance of P. issikii. Further studies are needed to determine their effect on the abundance of this host.

The factors that determine the number of parasitoid species parasitizing an invasive host have been studied by Cornell & Hawkins (1993).They account for the development of parasitoid assemblages in terms of the effects of residence time, biological characteristics of the introduced species and the host spectrum of the native parasitoids.Similar temporal changes in the species composition of the parasitoids attacking P. issikii is to be expected.One important biological characteristic of the host in this respect is its phenology.Grabenweger (2004) found that while parasitoid adults are active in the spring, C. ohridella larvae at that time are not suitable for attack.Therefore, the effect of the parasitoid on this leafminer's first generation is very low.According to Marković & Stojanović (2012), the percentage parasitism of the first generation of P. platani is also low.Similarly for M. robiniella the percentage parasitism of the first generation larvae is also a low (Csóka et al., 2009).In the case of P. issikii, larvae of both generations are very similar in terms of the timing of their development to the native Phyllonorycter species.However, studies indicate that native parasitoids are not able to regulate the populations of this recently arrived and established species.
Šefrová (2002) reports that the rate of development of the summer generation of P. issikii is strongly temperature dependent.Warmer temperatures result in faster development.Yefremova & Mishchenko (2008) predict that the warming climate may allow a third generation to develop.Similar effects associated with climate change are recorded for other insects (Thomson et al., 2010).
The dataset for Hungary resulted from our own 4-year rearing project (2011)(2012)(2013)(2014).We collected only mature leaf mines of P. issikii from 19 localities throughout Hungary (Szőcs et al., 2014;unpubl.).The mines were cut from the leaves and after a short period of drying were put in air ventilated plastic tubes (in order to avoid the problems with mould) for individual rearing.All the literature data cited in the present work were obtained from similar rearing of individuals and therefore considered comparable.
The parasitoids that emerged were kept in ethanol until identified.We have retained samples for further genetic studies.The results obtained during the first three years are published (Szőcs et al., 2014).
The Shannon diversity index was calculated in order to compare the parasitoid complexes of different hosts, as Matošević & Melika (2012) did in their paper.In order to estimate the significance of the differences in the diversity indexes, we used a twosample independent t-test.
rEsuLts And dIscussIon the species composition of the complex The presence of the lime leaf miner on the Eurasian continent dates back to the 1970-s.After it spread rapidly local native natural enemies began to parasitize the lime leaf miner.Based on recently published papers 46 parasitoid species have been reared from this leaf miner.A list of the species is presented in Table 1.Forty two of these species belong to the superfamily Chalcidoidea and four to the Braconidae.Members of the Eulophidae are generally associated with leaf miners, especially those of the genus Phyllonorycter.
In Europe and russia Tilia cordata is the primary host of P. issikii (Szabóky & Csóka, 2003;Ermolaev, 2014;Kirichenko, 2014).In natural conditions, it grows as a component of mixed broadleaved forests (Pigott, 1975).Therefore, it might be easy for the native parasitoids to shift to parasitize the novel host.
A summary of its spread is given by Ermolaev (2014).outside its native range the moth was first found in Korea (Kumata et al., 1983).In russia (Primorsky territory) it was first observed on Tilia amurensis (ruprecht, 1869) and Tilia mandshurica (ruprecht et Maximowicz, 1856) (Ermolaev, 1977), both of which are native plants.It was found developing well on Tilia cordata (Miller, 1768) in 1982(Yefremova & Mishchenko, 2008).According to Ermolaev (2014), in 1985 the moth reached Moscow.He describes two possible ways in which it might have been introduced into this region: cargo/transport (when the adults were hibernating) and the introduction with the host plant (as ornamental trees planted in parks).
The main damage it causes is considered to be primarily aesthetic, but according to Ermolaev & zorin (2011) high infestations can result in a decrease the nectar production of lime trees, causing serious economic loss to the honeybee keepers in russia (Ermolaev & zorin, 2011) .
P. issikii may have a significant negative effect on the herbivore guild of Tilia.As it has become by far the most dominant herbivore species feeding on lime (Segerer, 2008;Jurc, 2012), in many places it may eventually outcompete the native leaf miner species Stigmella tiliae (Frey, 1856) and Bucculatrix thoracella (Thunberg, 1794).

MAtErIAL And MEthods
We summarized the data in the available literature and two online databases containing host-parasitoid associations: the Universal Chalcidoidea Database hosted by the British Natural Histo-  (Csóka et al., 2009).
It is generally accepted that new resources (i.e. a new host) are primarily exploited by generalist species, which may be more flexible in their host searching behaviour and/ or adaptive potential and therefore more able to rapidly exploit a novel host.For example, S. gordius is known to be a generalist parasitoid with a broad host range.It is recorded as the dominant regulator of the lime leaf miner in ukraine by Mey (1991), in the Middle Volga basin in russia by Yefremova & Mishchenko (2008) and Yefremova et al. ( 2009) and as a secondary parasitoid by Ermolaev et al. (2011).In Hungary, it was the third most dominant species.The larva of this species is a solitary (only one adult emerges from a mine) larval-pupal ectoparasitoid (Mishchenko & Yefremova, 2012).The female lays an egg close to the host in a mine (Dowden, 1941).After the larva hatches, it kills the host and feeds on its haemolymph.The parasitoid pupa overwinters in the mine (Dowden, 1941).As a syn-ovigenic species, the adult matures eggs during its life time (Jervis et al., 2001(Jervis et al., , 2003;;Ellers & Jervis, 2004), therefore the protein resource obtained from the early stage leafminer larvae is important for the development of eggs (host feeding and stinging behaviour) (Jervis et al., 2008).This behaviour is very common among parasitoids (Askew & Shaw, 1974;urbaneja et al., 2002;Pinto et al., 2005;Bernardo et al., 2006).Similar behaviour is recorded for P. soemius, which is a dominant parasitoid with a significant effect on this host in russia (Ermolaev et al., 2011).This parasitoid is also a primary (sometimes secondary) solitary ectoparasitoid with a broad host range.According to Bernardo et al. (2006) this species is predator-like in its behaviour: the adult often kills (by stinging) host larvae without feeding (host-feeding) or ovipositing in the mines and its larvae even migrate from one mine to another (Bernardo et al., 2006).Such behavior is hard to quantify in the field.These traits make P. soemius one of the most important population control agent in agricultural pest management (Bernardo et al., 2006).H. geniculatus is another dominant species of solitary ectoparasitoid (Ermolaev et al., 2011), which overwinters as a pupa (Brockerhoff & Kenis, 1996).This parasitoid was reared only from russian samples (Ermolaev et al., 2011(Ermolaev et al., , 2013)).C. lynchus, does not play an important role in the regulation of P. issikii, but is recorded emerging from this host in almost all the studies carried out in Europe and russia.C. lyncus is a solitary ectoparasitoid, with the ability to behave as a secondary parasitoid.The eggs are laid on the host and the larvae overwinter (urbaneja et al., 2002).In Hungary (Szőcs et al., 2014), andBulgaria (Tomov, 2009) as well as in russia (Yefremova et al., 2009), M. frontalis is also one of the main controlling agents of this leaf miner.This species is a gregarious (more than one egg is laid in a mine) ectoparasitoid, whose larvae can behave like a primary, secondary or tertiary parasitoid.The female lays eggs near the host (Yefremova & Mishchenko, 2012) and it overwinters as a last instar larva (Lupi, 2005).
The parasitoids of the invasive P. issikii (just as in the case of other invasive species) include more idiobiont than koinobiont species.The more specialized koinobiont parasitoids need to adapt to the novel host's immune system (Pennacchio & Strand, 2006).Therefore, the evolution of a koinobiont parasitoid complex takes longer.This may explain why only a few koinobiont parasitoids emerged from the Hungarian samples of P. issikii.In contrast, the parasitoid assemblages associated with native species of leaf miner tend to include more endoparasitoid species (Askew & Shaw, 1979b;Sato, 1990;Szőcs et al., 2013;Yefremova & Kravchenko, 2015).In russia, where P. issikii has been present for longer, C. laomedon is an important koinobiont parasitoid (Yefremova & Mishchenko, 2008;Ermolaev et al., 2011;Yefremova et al., 2013), being ranked the third most dominant at every location.As a koinobiont (host remains alive after it is parasitized) the adult lays an egg on the cuticle of a leaf miner larva.After hatching, the larvae leave the caterpillar and start to feed as an ectoparasitoid (Yefremova & Mishchenko, 2012).This species is a primary parasitoid of a large number of leaf miners (De Prins & De Prins, 2014;Noyes, 2015).
The proportion of and diversity of ecto-and endoparasitoid species might be a good indicator of the degree of integration of a non-native species into the local fauna.There is no difference in the numbers of ectoparasitoid species parasitizing native and invasive species [based on our own records and those of Csóka et al. (2009)].In the case of P. issikii, the number of ectoparasitoid species is higher than the number of endoparasitoid species (14 ectoparasitoid species to 7 endoparasitoid species) (Szőcs et al., 2014).Two other invasive species (M.robiniella and P. robiniella) are also parasitized by a lower number of endoparasitoid species than native leaf-miners of the genus Phyllonorycter.Shannon diversity indices (Table 3) indicate that the diversity of the ectoparasitoid complex of the invader differ greatly from that of the native species (t = 5.6569, p = 0.0299, p < 0.05; for the two groups native/invasive).
The first native parasitoids to parasitize the novel host were most likely the species with a large host range, which encounter other species of leaf miners of the same genus, Phyllonorycter, on adjacent trees.Similar patterns of recruitment of host guild-associated parasitoids are recorded for invasive insect herbivores, for example the cynipid gall wasps on oak (Stone et al., 1995;Schönrogge et al., 1995Schönrogge et al., , 1996) ) and chestnut (Aebi et al., 2007) in Europe.

Percentage parasitism
Generally the incidence of attacks by parasitoids and predators on invasive species of herbivorous insects is low, at least initially (Schönrogge et al., 1995(Schönrogge et al., , 2011;;Grabenweger, 2004;Csóka et al., 2009;Marković & Stojanović, 2012;Matošević & Melika, 2012).The reasons are very simple.Initially, the local parasitoid fauna does not "recognize" the invasive species as a possible source of food.Alternatively, the invasive species may have a different life cycle such that native parasitoids are unable to detect the presence of this new food source.Examples include Cameraria ohridella (Grabenweger, 2004) and Parectopa robiniella (Csóka et al., 2009).In this case, the phenology of the native parasitoids needs to change if they are to track the novel hosts (Cornell & Hawkins, 1993).
The life stages and phenology of Phyllonorycter issikii are rather similar to those of native Phyllonorycter species.Therefore, it should be easier for the native parasitoids to adapt to this novel host than in case of C. ohridella and P. robiniella.Csóka et al. (2009) report that the percentage parasitism of P. robiniella in Hungary (0.3-15.3%) is much lower than that of M. robiniella (varied between 0.3-47.6%)and that this difference is possibly due to differences in their life cycles and phenology and absence of a closely related species of Parectopa.
The percentage parasitism of P. issikii varies between 0.9% and 37.2% (Table 4) across the area it has recently colonized.These low levels of percentage parasitism indicate that this species is not yet well integrated into the local food-web.Clearly, the effect of native parasitoids is limited, as is the case for other invasive species.
Mortality of leaf miners caused by parasitoids acting as predators is very common, since not only the larvae, but also the adults, feed on host larvae (host feeding, maturation feeding).This behaviour is common among synovigenic species of parasitoids, because they need extra protein to produce more eggs (Flanders, 1950;Godfray, 1994;Jervis et al., 2001;Harvey et al., 2013).The adults attack the first 1-3 instar larvae of Phyllonorycter.In fact, more Phyllonorycter are killed as a result of host-feeding by adult parasitoids than by parasitism (Askew & Shaw, 1979a, b).After host-feeding, female wasps' most likely search for oviposition sites close to the feeding location because on heavily infested leaves (mainly in the second generation), mines in different developmental stages can be found.The mortality caused by adult parasitoids as a result of their "maturation" feeding is usually not included in estimates of the percentage of hosts killed by these parasitoids.Therefore, the regulating potential of these ectoparasitoids is likely to be considerably greater than previously considered.This is likely to be the most important group of parasitoids of the lime leaf miner, particularly in the early stages of colonization.

tabLe 1 .
The species of parasitoids that have been recorded attacking P. issikii in the different areas it has recently colonized.uKr -ukraine, ruS -russia, JAP -Japan, BuL -Bulgaria, Cro -Croatia and HuN -Hungary.

tabLe 2 .
The dominant species of parasitoids of P. issikii recorded in the different areas it has recently colonized.• -dominant species, •• -secondary dominant species, ••• -tertiary dominant species.The dominance levels are those cited in the original papers.

tabLe 3 .
Comparison of the diversity of endo-and ectoparasitoids recorded attacking native and non-native leaf miners in Hungary based on the Shannon diversity index and our own data.* Szőcs et al., 2014; unpubl.; ** Csóka et al., 2009; *** Szőcs et al., unpubl.

tabLe 4 .
The percentage parasitism of P. issikii recorded in the different areas it has recently colonized.