J. De Prins et al.: Pupal morphology of Cameraria ohridella compared with Phyllonorycter 145

Anz. Scha¨dlingskunde ⁄ J. Pest Science 76, 145–150 2003, Springer Verlag ISSN 1436-5693

1Royal Museum for Central Africa, Tervuren, Belgium; 2University of Amsterdam, Zoological Museum, Section Entomology, Amsterdam, The Netherlands The pupal morphology of Cameraria ohridella compared with that of the Phyllonorycter (: )

By J. De Prins1,W.De Prins2 and E. De Coninck1

Abstract expansion, the parasitoid guild as well as the genetics The pupal morphology of the pest species Cameraria and the chemical communication are relatively well ohridella is described and compared with that of the genus known. An extensive bibliography can be found, among Phyllonorycter. The most striking differences are the longer others, in: De Prins and Puplesiene (2000) and antennal appendages, the absence of a cremaster, the De Prins and De Prins (2001). Papers on the mor- presence of strong lateral thorns on abdominal segments phology of the pre-imaginal stages are far less common. ˇ 1–5 and an area without small spines on the same abdominal Sefrova´ and Skuhravy´ (2000) described the larva of C. segments in Cameraria. ohridella and compared it with the larval morphology of three species of the genus Phyllonorycter, i.e. P. leuco- graphella (Zeller, 1850), P. medicaginella (Gerasimov, 1 Introduction 1930), and P. robiniella (Clemens, 1859) all from the In 2002, the very rapidly spreading Cameraria ohrid- region of Brno (Czech Republic). In this paper, we ella Deschka & Dimic´, 1986 reached as far west as the describe the morphology of the pupa of C. ohridella and British Isles (Agassiz, pers. comm.) after an incredibly compare it with the morphology of the pupae of fast invasion of most parts of Central and Western Phyllonorycter platani (Staudinger, 1870) and P. robini- ˇ Europe (Sefrova´ and Lasˇtu˚ vka, 2001; Guichard and ella (Clemens, 1859). Augustin, 2002). It is a pest of the horse chestnut tree (Aesculus hippocastanum L.) where it mines the upper 2 Material and methods leaf surface, and it can occur in such large numbers that almost the whole leaf area is covered with mines causing Leaf mines of C. ohridella were collected form Aesculus the leaves to drop as soon as August. C. ohridella is the hippocastanum L. at Tervuren (Belgium, Province of Vlaams- only known species of the genus Cameraria Chapman, Brabant) in the summer of 2002. Tenanted mines of P. platani 1902 to occur in Europe. The genus has 7 representatives were collected at Antwerpen-Deurne (Belgium, Province of in Central and East Asia (Kumata, 1963; Puplesis et al., Antwerpen) on Platanus orientalis L. and those of P. robiniella 1996) but it is common in the Nearctic region where 39 were sampled at Mol (Belgium, Province of Antwerpen) from species have been described (Davis, 1983). It is very Robinia pseudoacacia L., both in the autumn of 2002. The leaf closely related to the genus Phyllonorycter Hu¨ bner, 1822. mines were put in Petri dishes at room temperature allowing Thus far, no effective control measures have been the caterpillars to pupate. The pupae were subsequently developed against C. ohridella, though many chemical extracted from the leaf mines and directly processed for use treatments have been applied (Blu¨ mel and Hausdorf, under the scanning microscope. 1997; Krehan, 1997a; Perny, 1997), including for The movable pupal abdominal segments were immobilized example the injection of Confidor into the tree trunk with momentary heat. Pupae were sputtered with gold using (Feemers, 1997; Krehan, 1997b). Natural parasitoids of a Bal-TEC/SCD 005 Sputter Coater. The images were taken C. ohridella have not spread together with the species, with a Jeol MP 35060 camera combined with a Jeol JSM-5400 but many Eulophidae and some Eupelmidae, Pteromal- LV Electron Scanning Microscope and processed using the idae and Ichneumonidae that parasitize several Phyllon- Orion 4 High Resolution Image Grabbing System software. orycter species have been bred from C. ohridella Five specimens of Cameraria ohridella and three specimens of (Hellrigl, 1999; Freise et al., 2002). However, none Phyllonorycter platani and Ph. robiniella were examined with of these species seem able to really control the mass this method in great detail, while another ten specimens occurrence of the : the parazitation rate is slowly of each species were studied under a dissecting microscope increasing, from almost 0% in 1999 to 10% in 2002 in (60·). Belgian breeding experiments and up to almost 30% in Polish populations (Buszko, pers. comm., 2003). The 3 The pupal morphology of Cameraria ohridella population dynamics of C. ohridella can be effectively monitored with a synthetic pheromone (Kindl et al., In many cases, pupae of Phyllonorycter show enough 2002). The most effective control measure in parks and interspecific differences to enable their identification by lanes until now has been the systematic removal of fallen pupal characters only. Most useful are the characters leaves and their subsequent burning. situated on the dorsal and lateral surfaces of the Much attention has been paid lately to the biology, abdominal segments (Wilson, 1985). The overall exter- distribution and biological control of Cameraria ohrid- nal morphology of the pupa of C. ohridella closely ella. Also the morphology of the adult, its recent resembles that of the species in the genus Phyllonorycter.

DOI 10.1007/s10340-003-0009-2 146 J. De Prins et al.: Pupal morphology of Cameraria ohridella compared with Phyllonorycter

Fig. 1. Cameraria ohridella, pupa in dorsal view. Scale bar ¼ 500 lm. Fig. 2. Cameraria ohridella, pupa in lateral view. Scale bar ¼ 500 lm. Fig. 3. Head of the pupa of Cameraria ohridella, dorsal view showing the pointed beak which serves as a ÔÔcocoon-cutterÕÕ. Scale bar ¼ 50 lm. Fig. 4. Head of the pupa of Cameraria ohridella, lateral view of head showing the downwards pointed beak with upwards curved tip. Scale bar ¼ 100 lm. Fig. 5. Basis of future wings of Cameraria ohridella showing the finger-like structure. Scale bar ¼ 10 lm. Fig. 6. Dorsal part of abdominal segments 4–5 of pupa of Cameraria ohridella with anterior and posterior rows of small spines and almost equal central part. Scale bar ¼ 50 lm.

However, there are also some clear differences which free distally. The antennae are longer than the hindlegs will be pointed out in this chapter. The general form is (figs. 1, 2), while in Phyllonorycter species the opposite elongated oval, cylindrical, gradually narrowing in the situation applies (Gregor and Patocˇ ka, 2001) (fig. 13). last five segments as can be seen in dorsal view (fig. 1), The anterior end of the head capsule is furnished with a and a little flattened as can be seen in lateral view (fig. 2). sharply pointed beak, slightly curved ventrally, with The colour varies from light to dark brown as in most which the pupa cuts through the cocoon just prior to the lepidopterous species. emergence of the adult (figs. 3, 4). This beak has a The head is of triangular form without apparent triangular shape when observed from above, with sides appendages except for the antennae which extend well making an angle of just over 90 (fig. 3). In lateral view, beyond the last abdominal segment and are completely the beak makes an angle just under 90 and is pointed J. De Prins et al.: Pupal morphology of Cameraria ohridella compared with Phyllonorycter 147

Fig. 7. Dorso-lateral part of abdominal segments 4–5 of pupa of Cameraria ohridella with strong hook and additional sensilla on each segment. Scale bar ¼ 50 lm. Fig. 8. Abdominal segments 9–10 of pupa of Cameraria ohridella with two dorsal and two ventral tubercules. Scale bar ¼ 50 lm. Fig. 9. Head of the larva of Cameraria ohridella,4th instar, dorsal view of tip of head with labrum. Scale bar ¼ 50 lm. Fig. 10. Head of the larva of Cameraria ohridella,5th instar, ventral view of tip of head, showing labium and spinneret. Scale bar ¼ 50 lm. Fig. 11. 5th instar of Cameraria ohridella, thoracic leg with rudimentary claw. Scale bar ¼ 10 lm. Fig. 12. 5th instar of Cameraria ohridella, abdominal proleg with 13 crochets arranged in a semi-circle. Scale bar ¼ 10 lm.

ventrally except for the very point which is turned up spines along its anterior margin (fig. 2). At the base of slightly (fig. 4). the future wings, there is a structure of long, finger-like The thoracic segments bear the appendages for the teeth, resembling a comb, situated opposite a narrow future legs and wings. Those of the hind and middle legs row of raised chitinous bulbs (figs. 4–5). The whole area extend almost as far beyond the abdominal tip as the of the head capsule is covered with round or slightly antennae, those of the front legs do not reach the oval holes enabling the exchange of air (fig. 5). abdominal tip, and those of the wings reach the 6th or 7th The abdominal segments 1–6 have a strongly chiti- abdominal segment (fig. 1). The prothoracic segment nized plate on the dorsal side, each of which possesses a possesses two spines placed just laterally of the narrow band of small, tooth-like shaped asperites at the central plate close to the head capsule. They are directed anterior margin and a similar band of gradually becom- anteriorly and a little outwardly. Similar spines occur on ing larger asperites at the posterior margin. The largest the meso and metathoracic segments, but here these ones are oval in shape and bluntly pointed (fig. 6). The spines are directed posteriorly. The metathoracic seg- central plate of the 6th segment is completely covered ment, moreover, possesses a narrow band of very small with small asperites, while the central area of segments 148 J. De Prins et al.: Pupal morphology of Cameraria ohridella compared with Phyllonorycter

Fig. 13. Phyllonorycter robiniella, pupa in ventral view. Scale bar ¼ 500 lm. Fig. 14. Phyllonorycter robiniella, last abdominal segments with long sensillae. Scale bar ¼ 50 lm. Fig. 15. 3rd abdominal segment of pupa of Phyllonorycter robiniella with row of strong dorsal hooks. Scale bar ¼ 10 lm. Fig. 16. Phyllonorycter platani, pupa in dorsal view. Scale bar ¼ 500 lm. Fig. 17. Abdominal segments 6–7 of pupa of Phyllonorycter platani with long sensillae and completely covered area with small spines. Scale bar ¼ 100 lm. Fig. 18. 10th abdominal segment of pupa of Phyllonorycter platani with cremaster and two pairs of equal-sized hooks. Scale bar ¼ 50 lm.

1–5 is devoid of them (fig. 6). Moreover, at their lateral Abdominal segments 7–9 are dorsally completely cov- margins, each of these segments 1–5 possesses a strong, ered with small spines pointing to the posterior end. inwardly curved thorn and a spine, similar to that on the Segment 7 possesses 2 short spines pointing posteriorly. thoracic segments (fig. 7). The 6th segment only posses- The 9th abdominal segment is small and possesses two ses the spine. These strong thorns apparently serve to small dorsal tubercules protruding laterally. The 10th anchor the pupal skin while the moth emerges abdominal segment has not the characteristic cremaster (Deschka and Dimic´ , 1986). A few Quercus -feeding as in many lepidopteran species, which is also present in Phyllonorycter species, notably P. muelleriella (Zeller, most species of the genus Phyllonorycter (fig. 18). 1839) and P. messaniella (Zeller, 1846), possess similar Instead, that segment possesses two short, rather blunt thorns at the lateral margins of abdominal segments 1–3; tubercules on the ventral side, laterally of the anellus, in P. quercifoliella (Zeller, 1839) similar but smaller curved downwards and pointing anteriorly (fig. 8). thorns are present (Wilson, 1985, fig. 105u–w). These structures probably have not the same function as J. De Prins et al.: Pupal morphology of Cameraria ohridella compared with Phyllonorycter 149

Table 1. Differences in the pupal morphology between the genera Phyllonorycter and Cameraria.

Phyllonorycter Cameraria ohridella Appendages for future legs and antennae Extending till abdominal segments 6 or 7 Extending beyond abdominal tip. Appendages for future antennae Shorter than appendages for future legs Longer than appendages for future legs Strong lateral thorns on abdominal Absent(1) Present segments 1–5 Cremaster Present(2) Absent Dorsal plate of abdominal segments 1–5 Completely covered with small asperites Not completely covered with small asperites (1)Similar thorns are present on the abdominal segments 1–3 in P. muelleriella, P. messaniella and smaller thorns in P. quercifoliella. (2)A cremaster is absent in very few Phyllonorycter species, e.g. P. helianthemella, P. robiniella.

the hooks or spines on a cremaster, since no such similar the future hind legs (fig. 13). Also the hairlike, lateral structures were observed, except perhaps for two small and dorso-lateral setae on the thoracic and abdominal crochets at the very tip of the two dorsally placed segments are much longer and more conspicuous in the tubercules. species of the genus Phyllonorycter (figs. 13, 16), while they are much shorter in Cameraria and almost spine- like on the thoracic segments (fig. 2). 4 Additional notes on the larval morphology of The pupa of Phyllonorycter robiniella does not show Cameraria ohridella the typical cremaster as in other species of the genus Though the larval morphology of C. ohridella has Phyllonorycter (fig. 14), a character which it shares with ˇ already been extensively described by Sefrova´ and P. helianthemella (Herrich-Scha¨ffer, 1860) (Gregor Skuhravy´ (2000), some important details have not been and Patocˇ ka, 2001). On the other hand, it possesses illustrated with scanning microscope photographs but some strong hooks on, e.g., the third abdominal segment merely with schematic drawings. (fig. 15) which probably have the same function as the As in most Gracillariid species, there are clear-cut hooks on a cremaster. The pupal skin on the abdominal morphological differences between the sap-feeding and segments of Phyllonorycter species is almost completely the tissue-feeding instars in C. ohridella. As pointed out covered with small spines, most often gradually dimin- by Skuhravy´ (1998) there are 4 sap-feeding and 2 tissue- ishing in size from the anterior to the posterior edge of feeding instars in C. ohridella. In a sap-feeding instar of each segment (figs. 14–15, 17). this species, the anterior margin of the labrum is The pupa of Phyllonorycter platani does show the serrated, with 2 or 3 large, sharp teeth on the outer typical cremaster as in the other Phyllonorycter species sides, much resembling the cutting edge of a saw, (fig. 18). Furthermore, the dorsal side of all abdominal enabling the labrum to cut the cell walls close to the segments are completely asperate, with smaller asper- cuticle when the larva moves its head to and fro, as is ites along the anterior margin, and some bigger ones splendidly described by Kumata (1978) and by Emmet, posteriorly. This character state is common in many Watkinson and Wilson (1985). The labrum possesses other Phyllonorycter species, e.g. P. salicifoliella 6 round sensillae at either side of the centre, arranged in (Chambers, 1875) (Watson, 1956), but it is a striking two more or less parallel oblique rows (fig. 9). While the difference from the pupa of Cameraria ohridella, head morphology in the first 4 instars remains almost where the central area of these abdominal segments identical throughout and comprises mainly an increase is devoid of asperites (fig. 17). The cremaster bears in size, the transition to the 5th instar involves a radical two pairs of conspicuous hooks with which the pupa change in the morphology including, among other is anchored to the silken threads of the cocoon, things, the disappearance of the saw-like labrum and enabling the adult moth to escape from the pupal skin the appearance of a spinneret (fig. 10). during emergence. These hooks are of almost equal In the 5th and 6th instars, the thoracic legs are present size and placed at equal distances from each other. In but, in contrast with the genus Phyllonorycter, they are some cases, the distal ends of the inner hooks are not much reduced and only preserved in the form of a small curved (Gregor and Patocˇka, 2001). This condition ˇ rounded tuber with a rudimentary claw (Sefrova´ and was not observed in any of the Belgian specimens. Skuhravy´, 2000) (fig. 11). The ventral prolegs on Differences in the pupal morphology between the abdominal segments 3–5 and the anal prolegs are equally genera Phyllonorycter and Cameraria are summarized reduced and provided with 12–20 crochets arranged in table 1. around a central opening in a semicircle (fig. 12). Future study should reveal whether these character states in the pupa of Cameraria ohridella are also present in the other Cameraria species, of which most 5 Discussion have been described from the Nearctic region (Davis, In most species of the genus Phyllonorycter, the 1983). thoracic appendages are shorter than the pupa and do usually not reach beyond the 6th or 7th abdominal Acknowledgements segment (fig. 13). The most striking difference with the genus Cameraria is found in the shorter antennal We would like to thank Dr. M. De Meyer (Tervuren) appendages when compared with the appendages of for valuable comments after critical reading of the 150 J. De Prins et al.: Pupal morphology of Cameraria ohridella compared with Phyllonorycter manuscript, Dr. J. Buszko (Torun) for information on Krehan, H. (1997b): Erste Erfahrungen mit Bauminfusionen the occurrence of C. ohridella in Poland and Dr. D. J. L. gegen die Rosskastanienminiermotte. Forstschutz Aktuell 21, Agassiz (Gravesend) for information on the occurrence 26. of C. ohridella in the British Isles and comments on the Kindl, J.; Kalinova, B.; Freise, J.; Heitland, W.; Augustin, manuscript. S.; Guichard, S.; Avtzis, N.; Svatos, A. (2002): Monitoring the population dynamics of the Horse Chestnut Leafminer Cameraria ohridella with a synthetic pheromone in Europe. References Plant Protection Science 38, 131–138. Blu¨ mel, S.; Hausdorf, H. (1997): Versuche zur Kontrolle von Kumata, T. 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