Introduction

The leaf-miner agromyzid fly Chromatomyia horticola (Goureau 1851) has a cosmopolitan range of distribution, damaging crops and ornamental plants in many countries throughout the world (Anon. 1987; Dempewolf 2006; Spencer 1973; von Tschirnhaus 1969). It has highly polyphagous larvae and although listed to date by Benavent-Corai et al. (2005) from 230 species of the dicot and monocot genera of herbaceous plants, many additional plants do not appear on the list. Spencer (1990) presented a Table of 35 host plant families. Hosts in the Brassicaceae, Fabaceae and Asteraceae are dominant for C. horticola (Spencer 1973, 1976, 1989, 1990; Raj et al. 1995). The larval instars of C. horticola were described by Melis (1935) [as Phytomyza atricornis Meig., cf. Griffiths 1967:1-2], and further morphological characters also by Cohen (1936) and Allen (1958) (as P. atricornis, both could possibly also refer to the sibling species C. syngenesiae Hardy, cf. Griffiths 1967), as well as by M. Dempewolf (2001, Dissertation, Univ. Bielefeld, Germany). The species pupates internally at the end of the mine, with the anterior spiracles of the whitish puparium projecting through the plant epidermis (Spencer 1976). The whitish mine is linear or serpentine on both the upper and lower surface of the leaf.

In Europe and western Turkey the complex of parasitoids reared from C. horticola comprises 21 species of Eulophidae (Table 1). Species composition presents six species of Chrysocharis [Ch. entedonoides (Walker), Ch. gemma (Walker), Ch. nephereus (Walker), Ch. orbicularis (Nees), Ch. pentheus (Walker), Ch. pubicornis (Zetterstedt) (Hansson 1985; Rizzo & Massa 2002, 2004; Vidal 1997)]; six species of Diglyphus [D. crassinervis Erdös (Erdös 1958), D. chabrias (Walker) (Yefremova et al. 2011), D. isaea (Walker) (del Bene et al. 1993; Gençer 2005; Kumar 1985), Diglyphus minoeus (Walker), D. pachyneurus Graham (Gençer 2005), D. poppoea Walker (Rizzo & Massa 2002)]; and 2 species of Cirrospilus [C. vittatus Walker (del Bene 1989) and C. variegatus (Masi) (Rizzo & Massa 2002, 2004)] Hemiptarsenus ornatus (Nees) (del Bene 1989; Rizzo & Massa 2002), Neochrysocharis formosus (Westwood) (del Bene 1989; Rizzo & Massa 2002), Omphale stigma Goureau (Goureau 1851), Pnigalio soemius Walker, P. incompletus Bouček (Rizzo & Massa 2002), Pediobius metallicus (Nees) (Bouček, 1965; del Bene 1989; Civelek 2002; Gençer 2005; Rizzo & Massa 2002), Semielacher petiolata Girault (Massa et al. 2001; Rizzo & Massa 2002). The parasitoid complexes of 15 species of Agromyzidae (including C. horticola) from the Middle Volga Basin were recently studied (Strakhova et al. 2013; Yefremova et al. 2012).

Table 1 Parasitoid species reared from Chromatomyia horticola mines by different authors during 1958–2011.
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Taxonomically, we concur with the generic transfer of all world Chromatomyia Hardy, 1849 species to the genus Phytomyza Fallén, 1810, as recently published by Winkler et al. (2009). Without the provision of a detailed discussion of the male genitalia, the larval morphology, the specific mode of pupation, and the extensive published discussions and opinions on the generic/subgeneric status, the synonymization of Chromatomyia (so widely used in the multilingual agricultural world literature, including handbooks) all remains highly puzzling for taxonomists and applied entomologists.

The aim of the present work was to document the parasitoid complex of C. horticola. This paper, which summarizes our original data and earlier published information on the eulophid species, seeks to uncover the relationships between species composition of parasitoids, C. horticola, and its host plants, and to analyze the ratio of ecto- and endoparasitoids of C. horticola, with emphasis on their development in the puparium.

Materials and methods

The study was conducted in three adjacent locations in the Middle Volga Basin (Russia): (i) Ulyanovsk, left bank of the River Volga, Park (54°22'N, 48°32'E), (ii) Ulyanovsk Province, Dimitrovgrad (54°13'N, 49°36'E), and (iii) Ulyanovsk Province, village Lebjazhye, 90 km E of Ulyanovsk (54°06' N, 49°36'E) .

Chromatomyia horticola mines appeared from the end of April until September in a temperate zone in Russia, with a maximum number in July, which is the warmest summer month in the study area. Leaves with C. horticola mines were collected from ruderal herbaceous plants near roads, in gardens, and parks from May to September 2010. This period corresponds to the seasonal peak in Agromyzidae activity, with highest growth of their host plants and highest species richness of insects in general.

Leaves were collected every 3 days from the same territory. Typical mines of C. horticola on leaves of white ox-eye daisy, chickory lettuce, hollyhock, and dandelion are illustrated in Figures 1–4. The collected leaves with larvae were kept in the laboratory in 0.25-liter containers under room temperature of 20–22°С. Individuals emerging from C. horticola mines in the containers were collected using a pooter and immersed in 75% ethanol prior to mounting and identification.

Figs. 1–4
figure 1

Mines of Chromatomyia horticola on leaves of different host-plants: 1. Mine with puparium on Taraxacum officinale; 2. Mine with puparium on Alcea rosea; 3. Three linear mines and one serpentine mine on Lactuca tatarica; 4. Mine on Leucanthemum vulgare

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Samples were sorted using a stereomicroscope МС-2 ZOOM, and photographed using Canon Power Shot A 640. The reared Eulophidae were identified by the Russian authors. Identification keys for Diglyphus species are available for Europe (Yefremova & Shroll 1996), for Chrysocharis (Hansson 1985), for Pediobius (Bouček 1965), and for other species according to the key for the European part of Russia (Triapitsyn 1978) and the Far East of Russia (Storozheva et al. 1995).

A standard statistical method was used for analysis of the data. The two-way hierarchical clustering analysis with paired group was applied in order to estimate preference of parasitoids for particular plants. Similarities between objects were measured using the Gower Similarity Coefficient.

The material is deposited in the Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia) (ZISP).

Results

A total of 353 leaves with 379 mines were collected from 14 plant species belonging to three families: Asteraceae, Brassicaceae and Malvaceae. Most leaves housed only one mine; only 26 of the 353 leaves (6.8%) contained two mines.

The available data allow assessment of whether the species composition of the reared parasitoids is representative of the true species richness in the study area. The number of collected leaves per species of plant and the number of parasitoid species hatched from these samples is a classic example of the number of species collected and the number of samples taken (Magurran 2004). Here 90% of the parasitoid species were already represented in the first 102.4 ± 12.7 leaves, which is far less than the total of 353 leaves collected in our research.

A total of 38 specimens of C. horticola and 349 specimens of eulophids were reared. Altogether, the revealed species assemblage comprises 16 Eulophidae species belonging to seven genera and three subfamilies (Eulophinae, Entedoninae and Tetrastichinae). The number of specimens of each species is listed in Table 2. Seven species (Chrysocharis viridis, C. trifasciatus, D. pusztensis, M. frontalis, N. aratus, P. cassidae, and P. pectinicornis) are new parasitoid records for C. horticola.

Table 2 Number of parasitoid species reared from leaves with Chromatomyia horticola mines collected from the different host plants during 2010 in Russia.
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The number of specimens per species varied widely. Two dominant species, D. isaea (89 specimens) and P. metallicus (82 specimens), comprised about half (49%) of the total number of captured specimens, while fewer than ten specimens were found for many other species.

The collected parasitoids were subdivided into two ecologically different groups. Larvae of ectoparasitoids develop on the body of the host (externally), while larvae of endoparasitoids develop inside the host. Some of the pre-imaginal stages of both dominant (ecto- and endoparasitoid) species are shown in Figures 5–10. A fully developed endoparasitoid is almost equal in size to that of the host and occupies almost the entire volume of the body of its host except for the head capsule. In contrast, ectoparasitoids are always smaller than their host. Although several conspecific ectoparasitoids at different developmental stages might feed together on the body of a dipterous larva without competitive behavior, the competitive presence of two parasitoid species on or in the body of a host larva has also occasionally been observed.

Figs. 5–10
figure 2

Development of parasitoids on/in larva/pupa of Chromatomyia horticola: 5. Larva of C. horticola with first instar endoparasitoid larva of Chrysocharis sp. on Leucanthemum vulgare, 6. Puparium of C. horticola with last instar larva of Pediobius metallicus on Artemisia vulgare, 7. Puparium of C. horticola with endoparasitoid pupa of Pediobius metallicus on Artemisia vulgare, 8. Ectoparasitoid larvae of Diglyphus sp. on larva of C. horticola and endoparasitoid inside body on Artemisia vulgaris, 9. Three Diglyphus crassinervis larvae of different ages on larva of C. horticola on Callistephus chinensis, 10. Ectoparasitoid larva of Diglyphus sp. on larva of C. horticola removed from mine of Leucanthemum vulgare

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Ectoparasitoids were represented in our species assemblage by genera belonging to the Eulophinae and Tetrastichinae, with eight species and 205 specimens (58.7% of total). Endoparasitoids were represented by genera of the Entedoninae, with eight species and 144 specimens (41.3%).

The majority of the parasitoid specimens (88.5%) emerged in June and July. The number of ecto- and endoparasitoids in this period was similar, around 50% ± 12% each. However, the sex ratio within these groups differed: in ectoparasitoids there were 4.2-fold and 4.4-fold more females than males in June and July, respectively, whereas for endoparasitoids these values were only 1.2-fold and 1.5-fold.

For example, the dominant species, D. isaea, was characterized by 3–5-fold more females than males in summer: in June the ratio was 38 ♀/11 ♂, and in July 32♀/6♂. In the dominant endoparasitoid, P. metallicus, males outnumbered females by 25 ♂/18 ♀ in June and by 16♂/14♀ in July. In other words, 1.3-fold and 1.1-fold more males emerged than females (Fig. 11). These differences in sex ratio between the ecto- and endoparasitoids were probably not related to the plant species. On the small tumble-mustard (Sisymbrium loeselii), a plant of the cabbage family, the ratio for D. isaea in June was 26 ♀/6 ♂ and in July 14♀/5♂, whereas for P. metallicus in June it was 6 ♀/15 ♂ and in July 10♀/13♂.

Fig. 11
figure 3

Sex ratio in the ectoparsitoid Diglyphus isaea and the endopasitoid Pediobius metallicus throughout the 2010 summer season in Russia

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To determine whether ecto- and endoparasitoids preferred a particular species of plant, eight parasitoid species with more than ten emerged specimens were chosen. According to their preference for a particular host plant, the parasitoids were divided into two main branches, separated at the 0.6 level of Gower distance (Fig. 12). Species composition between these branches separated into the ecto- and endoparasitoids. One branch contains the endoparasitoids (N. formosus, C. trifasciatus, P. cassidae, P. metallicus), and the other the ectoparasitoids (P. soemius, D. isaea, D. poppoea, D. pusztensis). These two branches cover different ranges of plants. Endoparasitoids were found on all collected species of plants, while ectoparasitoids were apparently limited to only about half of them: namely, A. tomentosum, A. vulgaris, C. vulgare, L. tatarica, and C. sumensi.

Fig. 12
figure 4

Two-way clustering preference of parasitoids for particular plants. Similarity measured by Gower Similarity Coefficient; algorithm is paired group. Data transformed to presence (1), absence (0 = empty cell).

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Discussion

More than 21 species of eulophids (12 ecto- and nine endoparasitoids) are known as parasitoids of C. horticola in Europe (see Introduction). Of these, only 11 species were included in this study. In contrast to previous studies, we omitted species of the genera Cirrospilus and Semielacher because species of Cirrospilus have never been reared from C. horticola in the Middle Volga Region and species of Semielacher are not known at all from Russia (Yefremova 2002). Omphale stigma, which was reared in France 162 years ago (Goureau 1851) but never again, was also omitted from the list. The species Pnigalio soemius is considered a valid parasitoid of C. horticola and was reared from C. horticola in our study as well as in other studies (e.g. Bouček & Askew 1968). In contrast to these results, Gebiola et al. (2012) reared this species from lepidopterous hosts and considered it a cryptic species. Neochrysocharis formosus is also accepted as a valid species, as confirmed by molecular investigation by Tetsuya et al. (2011).

The dominant species of the parasitoid complex of Chromatomyia fuscula Zetterstedt was Diglyphus begini (Ashmead) (Hågvar et al. 2000) and of Agromyza frontella (Rondani) – Diglyphus species (Coote & Ellis 1986); consequently it is an ectoparasitoid species. Heinz & Parrella (1990) showed for D. begini that females oviposit "female-eggs" on a large host of Liriomyza trifoli (Burgess), while "male-eggs" are oviposited on smaller hosts. The size of the host larvae was not measured in our study, but the number of females was usually 3–5-fold greater than that of males.

Pediobius metallicus was not a common species in the parasitoid complex of C. fuscula (Hågvar et al. 1998) and it is mentioned in our study for the first time as a dominant species of Chromatomyia spp. in general. The sex ratio in the genus Pediobius observed for P. foveolatus reared from Cocinellidae, was revealed as 1♂:1.33♀ for field-collected parasitoids and 1♂:6.75 for laboratory-reared ones, with an average ratio of 1♂:3.29♀ (Stevens et al. 1977). In our study the sex ratio of P. metallicus was 1.3♂:1♀. The sex ratio of P. metallicus reared from C. horticola is presented for the first time.

The terms idiobionts and koinobionts as understood by Askew & Shaw (1986), are not included in this paper because these species of Eulophidae (in our list of the parasitoid complex of C. horticola) coincides with the concepts ecto- and endoparasitoids. The differences in the association of the ecto- and endoparasitoids with particular species of plants probably derive from the sensitivity of parasitoid larvae to environmental factors. Endoparasitoids develop inside the bodies of their host larvae and are therefore protected by the homeostasis of the larvae from the influence of environmental conditions; whereas ectoparasitoid larvae develop outside the host's body, thus being more exposed to the influence of environmental factors.

The dominant species Diglyphus isaea, like D. begini (Hågvar et al. 2000), is a very important regulating species and is widely used as an agent against agromyzid leaf miner pests in biological control programs. The species P. metallicus may also be considered in future as an agent against leaf miner pests.