J. Entomol. Acarol. Res. Ser. II, 42 (1): 39-45 30 April 2010 G.VIGGIANI, F. NUGNES Description of the larval stages of Dryokosmus kuriphilus Yasumatsu (Hymenop- tera: Cynipidae), with notes on their phenology Abstract - The larval stages of Dryokosmus kuriphilus Yasumatsu are described  and illustrated. The terminal-instar larva shows marked variation in the mandibular  asymmetry and in number and position of the respiratory spiracles. Notes on the  larval phenology are given.  Riassunto - Descrizione degli stadi larvali di Dryokosmus kuriphilus Yasumatsu  (Hymenoptera: Cynipidae), con note sulla loro fenologia. Vengono descritti gli  stadi larvali di Dryokosmus kuriphilus Yasumastsu. La larva matura mostra mar- cate variazioni nell’asimmetria mandibolare, nonché nel numero e posizione degli  spiracoli tracheali. Sono date notizie sulla fenologia larvale.  Key words: variations, respiratory spiracle, asymmetric mandibles. INTRODUCTION The gall wasp Dryokosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) is a  worldwide chestnut pest, which reproduces by parthenogenesis and develops one genera- tion a year. In recent years the species was recorded also in Italy, where it quickly spread  from Piedmont to the other main areas where chestnut is grown (Graziosi & Santi, 2008).  Several studies have been carried out on the bio-ecology, natural enemies and control of  the cynipid (Itô, 1967; Aebi et al., 2007; Ôtake, 1980; Rieske, 2007, Viggiani & Voto,  2009), but the detailed morphology and phenology of the larva still remain unknown.  MATERIALS AND METHODS During 2008-2009 the life-cycle of D. kuriphilus was studied in two Campania  locations (Calvanico, SA; Teano, CE). Samples of 100 galls were taken weekly from  June 2008 to September 2009. All types of larvae in the galls were recorded and, for  each sample, 20 larvae of D. kuriphilus were mounted on slide using a mixture of water  and glycerine. For each larval type 5 permanent slides were made using balsam-phenol.  Journal of Entomological and Acarological Research, Ser. II, 42 (1), 201040 Observations and images of morphological and anatomical larval traits were taken by   Axiophot microscope equipped with camera lucida and photocamera. For SEM micro- scopy the traditional method of dehydration in alcohol, critical-point drying and gold- sputter coating was utilized. Before making preparations, all larvae were measured.  Fifty couples of left and right mandibles were dissected and permanent slides were  made in order to study their anatomical variation. For the same purpose mandibles of  the terminal-instar larval skin were removed from the pupal site and examined. The vari- ation in number and position of the respiratory spiracles was recorded in 127 terminal- instar larvae mounted in a mixture of water and glycerin. At present, at least in Italy, no  inquiline cynipid was recorded in the galls of D. kuriphilus. The terminology follows  Nieves-Aldrey et al. (2005). RESULTS larval instars The present study shows that D. kuriphilus undergoes three instars in the larval  development.  First-instar larva (Fig. 1). Subglobular, with very small mandibles (Fig. 2) which  are subtriangular and apparently unidentate. No tracheal system was detected by the  external and internal examination of the larva. Length: 0.2-0.6 mm. This shape derives  from the typical cynipid egg, characterized by a suboval body and a long and thin stalk  (Fig. 3). The first-instar larva develops slowly from the summer (July-August) to the  late March-April of the next year. Intermediate instar larva (Fig. 4). Hymenopterifom, mandibles (Fig. 5) distally with  two teeth, without tracheal system. Length: 0.8-1.5 mm. The intermediate larva stage  appears in April-May and lasts less than one month.  Terminal-instar larva (Fig. 6). Hymenopteriform, with asymmetric mandibles and  a number of variable teeth (Fig. 7). Length: in average 2.3 mm (n = 127; SD = 0.4). In  frontal view the well-developed head capsule (Fig. 8) shows the antennal areas (Fig.  8, AA), a subtriangular clypeus (Fig. 8, C), followed by a transverse labrum. The man- dibles (Fig. 8, M; fig. 9) are only partially exposed from the mouth opening. Maxillae  subtriangular (Fig. 8, MX) with vestigial palpi (Fig. 8, MP), as on labium, which shows  a marked salivary opening (Fig. 8, LA). Respiratory system with 4-6 pairs of spiracles. The terminal-instar is the only larval stage present in the D.kuriphilus galls from  late April to end of May.  Variation. Remarkable variation in the morphology of mandibles and respiratory  system is recorded within the terminal-instar. Mandibles. The distal parts of the left and right mandible of 50 specimens were  examined. The most common type (Figs. 10-13) shows a strong external tooth, ventrally  concave, followed by an internal and laminar structure. This structure is differently  developed and a toothed margin is sometimes present. In other types (Fig. 14-15) the  dental part is represented by 2 sclerotized teeth. 41G.Viggiani, F. Nugnes: Larval stages of Dryokosmus kuriphilus, with notes on their phenology Figs. 1-7. Dryokosmus kuriphilus: first-instar larva, dorsal view (fig. 1), mandible (fig. 2), egg  (fig. 3), intermediate larva, ventral view (fig. 4), mandible (fig. 5), terminal-instar larva, lateral  view (fig. 6), mandible (fig.7).  Journal of Entomological and Acarological Research, Ser. II, 42 (1), 201042 Fig. 8-9. Dryokosmus kuriphilus: terminal-instar larva. Head capsule, frontal view (fig. 8), mouth  opening and mandible (fig. 9). AA = antennal areas; C = clypeus; L = labrum; LA = labium; LP  = labial palpus; M = mandible; MP = maxillary palpus; Mx = maxilla; SO = salivary opening;  V = vertex.  43G.Viggiani, F. Nugnes: Larval stages of Dryokosmus kuriphilus, with notes on their phenology Respiratory system. The variation in number and position of the respiratory spira- cles have been studied in 127 larvae. The observations are reported in table 1. Most of the larvae have 4 (44.9%) or 5 (33.8) pairs of spiracles, the first situated  on the mesothorax and the others starting from the second abdominal segment. The  extreme number of respiratory spiracles found were 3 and 6.  DISCUSSION AND CONCLUSION The Cynipoidea (Hymenoptera) include endoparasitoids and phytophagous species;  most of the latter group are gall makers. Although they are distributed worldwide, and  have been subject to years of investigation, our knowledge of pre-imaginal morphol- ogy is fragmentary and far from complete (Stojanova & Draganov, 2008). Particularly  lacking is the knowledge on the youngest larval stages. The shape of the first-instar  larva follows that of the egg. The egg of D. kuriphilus falls in the category with a long  and thin stalk and a stout body, typical of the Cynipidae (Vårdal et al., 2003). The first- instar larva of this species has a globular shape and is macroscopically indistinguishable  Fig. 10-15. Terminal-instar larva of Dryokosmus kuriphilus. Main types of dental parts of the  mandibles (dorsal view).  Journal of Entomological and Acarological Research, Ser. II, 42 (1), 201044 from the body of the egg, after the loss of the peduncle, as reported by earlier authors  (Itö, 1967). A very slight body segmentation and the presence of very small mandibles  characterize this instar.  The variation of the terminal-instar larva in the Cynipoid is remarkable, even be- tween sexes (Stojanova & Draganov, 2008). Most of them concern the shape and the  asymmetry of the mandibles. In a comparative study of the terminal-instar larvae of  Cynipoidea, Nieves-Aldrey et al. (2005) pointed out that head sclerites and mandibles  offer many characters of phylogenetic value. The general features of the terminal-instar  larva of D. kuriphilus are similar to those of other Cynipini (Cynipidae), but the notable  variation shown by the dental part of the mandibles, suggests a more extended study of  this trait in other species. The intraspecific variation in number and position of the respiratory spiracles found  in the terminal-instar larva of D. kuriphilus is certainly surprising and apparently un- recorded for other species. Probably this trait is connected to the fluid content in each  loculus of the gall. In general, the maximum number of tracheal spiracles is linked to  a dry larval habitat. Some terminal-instar larvae of parasitic Hymenoptera in a host  containing body fluid show a reduced number of respiratory openings (Viggiani, 1973;  Viggiani, 1984).  ACKNOWLEDGMENT  The Author thanks Dr. Hege Vårdal, Department of Systematic Zoology, Evolution- ary Biology Centre, Uppsala University, Sweden, for reviewing the manuscript.  Table 1. Variation in number and position of the respiratory spiracles in the terminal-instar larva of Dryokosmos kuriphilus.  N. spiracles Position Percentage 3 T2 A2 A3 0.8 3 T2 A2 A6 0.8 4 T2 A2 A3 A6 3.9 4 T2 A3 A5 A6 0.8 4 T2 A2 A3 A4 2.4 4 T2 A2 A4 A6 44.8 4 T2 A2 A4 A7 0.8 5 T2 A2 A3 A4 A6 33.8 5 T2 A2 A3 A5 A6 0.8 5 T2 A2 A4 A5 A6 6.3 5 T2 A2 A3 A4 A5  1.6 5 T3 A1 A2 A5 A6 0.8 6 T2 A2 A3 A4 A5 A6  2.4 Legend: A1 = first abdominal segment; A2 = second abdominal segment; A3 = third abdominal segment; A4  = fourth abdominal segment; fifth abdominal segment; A6 = sixth abdominal segment; seventh abdominal  segment; T2 = mesothorax; T3 = metathorax. 45G.Viggiani, F. Nugnes: Larval stages of Dryokosmus kuriphilus, with notes on their phenology REFERENCES aebi a., sCHönrogge k., Melika g., quaCCHia a., alMa a., stone g. n., 2007 - Native and  introduced parasitoids attacking the invasive chestnut gall wasp Dryocosmus kuriphilus.- Bull.  OEPP/EPPO, 37: 166-171.  graziosi i., santi F., 2008 - Chestnut wasp (Dryocosmus kuriphilus): spreading in Italy and new  records in Bologna province. - Bulletin of Insectology, 61 (2): 343-348.  itô y., 1967 - Population dynamics of the chestnut gall-wasp, Dryokosmus kuriphilus Yasumatsu  (Hymenoptera: Cynipidae) IV. Further analyses of the distribution of eggs and young larvae  in buds using the truncated negative binomial series. - Res. Popul. Ecol., 9: 177-191.  nieves-aldrey J. l., vårdal H., ronquist F., 2005 - Comparative morphology of terminal- instar larvae of Cynipoidea: phylogenetic implications. - Zoologica Scripta, 34 (1): 15-36.  ôtake a., 1980 - Chestnut gall wasp, Dryokosmus kuriphilus Yasumatsu (Hymenoptera: Cynipi- dae): A preliminary study on the trend of adult emergence and some other ecological aspects  related to the final stage of its life cycle. - Appl. Ent. Zool., 15(1): 96-105.  rieske J. k., 2007 - Success of an exotic gallmaker, Dryokosmus kuriphilus, on chestnut in the  USA: a historical account. - EPPO Bulletin, 37: 172-174.  stoJanova a. M., draganov M. M., 2008. - Life cycle of Aylax hypecoi (Insecta: Hymenoptera: Cynipidae), a gall inducer on Hypecoum ssp. (Papaveraceae). - Cent. Eur. J. Biol., 3(2):  199-204. vårdal H., saHlen g., ronquist F., 2003 - Morphology and evolution of the cynipoid egg  (Hymenoptera). - Zool. J. Linn. Soc. Lond., 139: 247-260. viggiani G., 1973 - Osservazioni morfo-biologiche sull’Azotus pulcherrimus Merc. (Hymenoptera:  Aphelinidae). Ricerche sugli Hymenoptera Chalcidoidea. XL. - Boll. Lab. Ent. agr. Filippo  Silvestri 30: 300-311. viggiani G., 1984 - Bionomics of the Aphelinidae. - Ann. Rev. Entomol. 29: 257-276. viggiani g., voto A., 2009 - Preliminary data on the phenology of Dryocosmus kuriphilus Yasu- matsu and the beneficial arthropodfauna of the chestnut in Campania. - Castanea 2009 - 1st  European Congress on Chestnut - 5° Convegno Nazionale Castagno. Cuneo, Italy, 13-16  October. Abstracts: 67.  proF. gennaro viggiani, Dipartimento di Entomologia e Zoologia Agraria “Filippo Silvestri”,  Via Università, 100 -80055- Portici (NA). E-mail: genviggi@unina.it Accepted 31 March 2010