J. Ent. Acar. Res. Ser. II, 42 (3): 171-181 30 December 2010 I. TóBIáS, F. Kozár, B. M. KaYDaN, K. FeTYKó Use of molecular tools for the identification of males of some scale insects (Hemiptera: Coccoidea), in pheromone traps used for monitoring and comparison with females Abstract - Species from Pseudococcidae family were studied. It was determined  that the dry males of Planococcus citri, and Pseudococcus comstocki, collected by  pheromone traps could be useful for the molecular analyses too. The ITS-2 sequences  of males and females in case of Pl.citri, Planococcus ficus and Ps. comstocki were  identical. This molecular method could differentiate the two mealybug species and  this method can be useful to have idea specimens collected by pheromone traps.  Riassunto - Uso degli strumenti molecolari per l’identificazione di maschi di cocci- niglie (Hemiptera: Coccoidea), in trappole a feromoni utilizzate per monitoraraggio e comparazione con le femmine. Sono state prese in considerazione specie appartenenti alla famiglia degli Pseudo- coccini. e’ stato visto che maschi essiccati di Planococcus citri, e Pseudococcus comstocki, raccolti da trappole a feromoni possono essere utilizzati anche per analisi  molecolari. La sequenza ITS-2 di maschi e femmine di P.citri, Planococcus ficus e  P. comstocki è identica. Questo metodo molecolare può differenziare le due specie  di cocciniglie e può essere utile per avere un’idea delle specie raccolte in trappole  a feromoni. Key words: Planococcus citri, Planococcus ficus, Pseudococcus comstocki, male,  pheromone trapping, climate change. The climate change already supported spread of new pest species, among them a  lot of scale insect species (Pseudococcus comstocki (Kuwana), Pseudococcus viburni (Signoret), Planococcus citri (risso), Planococcus ficus (Signoret), etc, which are very  important virus vectors in different crops. These species shows a substantial northward  spread in different parts of europe especially in the last forty years (Ben-Dov et al.,  2009; Kozár, 1997, 1998, 2005; Kozár and Nagy Dávid, 1986; Kozár and Szentkirályi,  2005; Pellizzari, 1991; Godinho and Franco, 2001; Sentenac and Kuntzmann, 2003;  Boudon-Padieu and Maixner, 2007; Sforza et al, 2005; etc).  The pheromone traps are useful and easy method to study on spread of these pests,  although they collect only males. The use of pheromone traps to monitor scale insect  Journal of entomological and acarological research, Ser. II, 42 (3), 2010172 populations requires some basic knowledge about morphology of males, but the identi- fication of scale insect males is a current problem because all descriptions and keys are  based only on females (Kosztarab and Kozár, 1988). Kozár et al. (1996, 1997) prepared  morphological and biological keys for males collected by pheromone traps of Diaspidi- otus (Quadraspidiotus) perniciosus (Comstock) because it was found that this pheromone  compounds are not species specific. This result was verified by molecular method by  Frey and Frey (1995). From this point of view the identification problems of the males of  Pl. citri, Pl. ficus, and Ps. comstocki taxa collected by pheromone traps could be solved  by using more detailed morphological analyses combined with molecular approaches. The aim of this work was (1) to present results of the monitoring of distribution three  mealybug species in different parts of europe, (2) to study the molecular differences of  scale insect males of two mealybug species collected by pheromone traps which can  occur together easily in the same pheromone traps, (3) to compare the sequences of  females and males in case of Pl. citri, Pl. ficus, and Ps. comstocki. MaTerIaL aND MeTHoDS Monitoring of mealybugs was conducted by Nagykovácsi type tent trap (10x10 cm),  by using (pheromone ingredients for Pl. citri; (+)-2,2-dimethyl-3-(1-methylethenyl)  cyclobutanemethanol acetate, for Pl. ficus; (S)-lavandulyl senecioate-(S)-lavandulyl  isovalerate), for Ps. comstocki; (2,6-dimethyl-l,5-heptadien-3-ol acetate) with Soveu- rode /Witasek Pflanzenschutz GmbH, austria/ glue and Biochemtech /Biochemtech Ltd.  Kishinev, Moldavia/ pheromone dispensers.  The traps were used in austria (Wien), Slovakia (Bratislava), Serbia (Belgrade),  Greece (athens, Iraklion, and Chania), Macedonia, and in Hungary in 2009 and 2010,  in different times and places (Tab 1).  Tab 1 - Number of mealybug males collected by pheromone traps in 2009*. Country, Locality Time period Planococcuscitri Planococcus ficus Pseudococcus comstocki Hungary, Budapest, Ördögárok ú.  04.28-07.31.2009 0 0 0 Hungary, Budapest, Ördögárok ú.  07.31-09.02.2009 8 2 1 Hungary, Budapest, Ördögárok ú.  09.02-10.02.2009 0 - 0 Hungary, Budapest, Ördögárok ú.  10.02-11.02.2009 3 - 2 Hungary, Budapest, M0,Csepel 05.18-03.08.2009 0 0 0 Hungary, Budapest, M0,Csepel 03.08-03.09.2009 2 0 0 Hungary, Budapest, M3, Szilas 05.11-07.30.2009 0 0 0 173I. Tóbiás et al.: Molecular tools for the identification of males of scale insects Hungary, Budapest, M3, Szilas 05.11-07.30.2009 0 0 0 Hungary, Budapest, M3, ecséd 05.11-07.30.2009 0 0 0 Hungary, Budapest, M3, ecséd 07.30-09.09.2009 1 0 0 Hungary, Budapest, M5, Kecskemét 05.14-07.28.2009 0 0 0 Hungary, Budapest, M5, Kecskemét 07.28-09.03.2009 0 0 0 Hungary, Budapest, M5, röszke 05.14-07.28.2009 0 0 0 Hungary, Budapest, M5, röszke 07.28-09.03.2009 0 0 0 Hungary, Budapest, M7, Budaörs 05.06-07.30.2009 0 0 0 Hungary, Budapest, M7, Budaörs 07.28-08.29.2009 1 0 0 Hungary, Budapest, M7, Velence 05.06-07.24.2009 0 0 0 Hungary, Budapest, M7, Velence 07.08-08.31.2009 2 1 1 Hungary, Budapest, M7, Töreki 05.06-07.25.2009 0 0 0 Hungary, Budapest, M7, Töreki 07.25-10.09.2009 3 1 2 Hungary, Budapest, M7, Letenye 05.06-07.25.2009 0 0 0 Hungary, Budapest, M7, Letenye 07.25-10.09.2009 0 - 0 Hungary, Nagykovácsi, around greenhouse 04.28-07.31.2009 22 0 0 Hungary, Nagykovácsi, around greenhouse 07.31-09.06.2009 95 1 2 Hungary, Nagykovácsi, in greenhouse 05.10-06.10.2009 8 0 0 austria, Wien 04.03-07.29.2009 0 0 0 austria, Wien 07.29-08.28.2009 3 0 0 Greecex, olympus Plaza 09.20-09.26.2009 0 - 0 Greece, Kifissia 09.20-09.26.2009 14 - 0 Greece, Iraklion 09.20-09.25.2009 48 - 41! Makedonia,  Vardar bridge 09.20-09.27.2009 0 - 0 Slovakia, Bratislava 07.29-08.28.2009 102 0 0 Switzerland, Lausane 06.16-06.28.2009 0 0 0 *  Between 04.04-04.10, 2010 the traps in Serbia (Beograd), Greece (Gevgelija, Thessaloniki, olympus plaza,  Kifissia, Chania), and in Hungary (Budapest) between 05.01-05.31, 2010, did not collected males of these  three species. Journal of entomological and acarological research, Ser. II, 42 (3), 2010174 DNA extraction, amplification, cloning and sequencing Total genomic DNa was extracted from single individuals (males and females)  using reDextract-N-amplTM Tissue PCr Kit (Sigma) according to manufacturer  instruction. Females and males of P. citri, P. ficus and P. comstocki were tested (Tab  2.). after preliminary experiments CaS5p8sFc (sense) (5’-gcgaacatcgacaagtcgaacg- cacat-3’) and CaS28sB1d (antisense) (5’-ttgttttcctccgcttattaatatgcttaa-3’) primer pair  (Kim and Lee 2008) was selected for PCr. The primers amplified the ITS 2 sequence  of nuclear DNa. PCr was performed using Taq DNa polymerase (Fermentas) in  thermo-cycler (eppendorf Mastercycler gradient) according to the following proce- dure: initial denaturation at 95 oC for 4 min, followed by 40 cycles of 95 oC for 30 sec,  annealing temperature 50 oC for 30 sec, extension at 72 oC for 60 sec; final extension  at 72 oC for 10 min. The PCr products were purified using Gel/PCr DNa Fragments  extraction Kit (Geneaid). Purified PCr products from 3 dry individuals of P. citri from pheromone traps, and three females living on Ficus benjamina (Budapest) and  3 females of P. comstocki from mass culture on potato (Van), and three males from  ethanol from mass culture on potato (Van). Pl. ficus individuals (2 females and 2 males)  were from mass culture on potato (Van). each were cloned into CloneJet (Fermentas)  vector and inserted into Escherichia coli DH5α competent cells. all cloning steps  were based upon standard molecular biology protocols (Sambrook et al. 1989). The  recombinant plasmids isolated from selected colonies were sequenced using pJeT1.2  forward and reverse primers, the PCr products of two P. citri, and P. comstocki were  sequenced by CaS5p8sFc and CaS28sB1d primers by an automated DNa sequencer  (applied Biosystem Gene analyzer 3100). Sequence comparisons were performed  using Wisconsin Package version 10.0 Genetic Computer Group (GCG) sequence  analysis software (Devereux et al. 1984). reSULTS aND DISCUSSIoN The pheromone trap catches show that only Pl. citri males were collected (Tab 1.)  and this species was caught only in Greece (Kifissia, Iraklion) in field conditions. In other  places such as in Hungary (Nagykovácsi), or Slovakia (Bratislava) the insects caught on  outdoor plants probable originate from greenhouses plants. In these places, this species  probably could not overwinter, or may be only in mild winter. The males collected by Ps. comstocki pheromone traps in Greece (Crete, Iraklion)  belongs to Pl. citri according to the molecular data, which was abundant in park on  Catalpa sp., Ficus sp., Morus sp. and other plants, and their males could appear ac- cidentally in Ps. comstocki traps.  on the other hand Pl. ficus and Ps. comstocki were not found in studied places and  times. The one or two males caught by traps in some places probable belong to other  genera and species.  175I. Tóbiás et al.: Molecular tools for the identification of males of scale insects Ta b 2 - O ri gi n an d ty pi ng o f m ea ly bu gs s tu di ed h er e. Sp ec ie s O ri gi n ty pe V oc he r no . G en eB an k ac ce si on n o. Se qu en ci ng P se ud oc oc cu s co m st oc ki Tu rk ey  V an  U ni ve rs ity  la bo ra to ry  c ul tu re fe m al e K oz ar F  N o.  9 25 4 hm 62 85 68 80 1  bp P se ud oc oc cu s co m st oc ki Tu rk ey  V an  U ni ve rs ity  la bo ra to ry  c ul tu re fe m al e K oz ar F  N o.  9 25 4 hm 62 85 69 80 1  bp P se ud oc oc cu s co m st oc ki Tu rk ey  V an  U ni ve rs ity  la bo ra to ry  c ul tu re m al e,  d ry K oz ar F  N o.  9 25 4 hm 62 85 70 80 1  bp P se ud oc oc cu s co m st oc ki Tu rk ey  V an  U ni ve rs ity  la bo ra to ry  c ul tu re m al e,  in  e th an ol K oz ar F  N o.  9 25 4 hm 62 85 71 80 1  bp P se ud oc oc cu s co m st oc ki K or ea fj 43 01 47 13 57  b p P se ud oc oc cu s co m st oc ki Fr an ce 09 00 00 67 gu 13 46 68 70 9  bp P se ud oc oc cu s co m st oc ki Fr an ce G u1 34 66 9 70 9  bp P s. co m -P C r 1 Tu rk ey  V an  U ni ve rs ity  la bo ra to ry  c ul tu re m al e,  e th an ol K oz ar F  N o.  9 25 4 73 9  bp P s. co m -P C r 2 Tu rk ey  V an  U ni ve rs ity  la bo ra to ry  c ul tu re m al e,  d ry K oz ar F  N o.  9 25 4 74 1  bp P la no co cc us c itr i Ir ak lio n,  C re te , P .c om st oc ki  fe ro m on e  tr ap m al e,  d ry K oz ar F  N o.  9 46 1 hm 62 85 72 77 3  bp P l.c itr i  P C r 1 H un ga ry m al e,  d ry K oz ar  F . N o  92 03 71 2  bp P l.c itr i P C r 2 H un ga ry m al e,  d ry K oz ar  F . N o  94 62 71 4  bp P la no co cc us c itr i H un ga ry ,  fe m al e K oz ar F  N o.  9 25 5 hm 62 85 73 77 3  bp P la no co cc us c itr i H un ga ry ,  fe m al e K oz ar F  N o.  9 25 5 hm 62 85 74 77 3  bp P la no co cc us c itr i Ir ak lio n,  C re te , P .c om st oc ki  fe ro m on e  tr ap m al e,  d ry K oz ar F  N o.  9 46 1 hm 62 85 75 77 3  bp P la no co cc us c itr i H un ga ry ,  fe m al e K oz ar F  N o.  9 20 4 hm 62 85 76 77 3  bp P la no co cc us c itr i K or ea fj 43 01 45 13 32  b p P la no co cc us c itr i Fr an ce 08 08 9i nr a gu 13 46 78  6 88  b p P la no co cc us c itr i Fr an ce 08 00 2i nr a gu 13 46 75 68 8  bp P la no co cc us m in or Fr an ce 08 03 22 3 gu 13 46 76 68 6  bp P la no co cc us fi cu s Fr an ce 08 25 58 in ra gu 13 46 77 69 2  bp P la no co cc us fi cu s Tu rk ey m al e K oz ar  N o9 58 7/ 1 77 0  bp P la no co cc us fi cu s Tu rk ey fe m al e K oz ar  N o9 58 7/ 2 77 0  bp Journal of entomological and acarological research, Ser. II, 42 (3), 2010176 Characteristics of sequences It was determined that the used DNa extraction procedure was suitable to handle  adequate quantity and quality DNa from dry males from pheromone traps (6-7 months  kept at laboratory condition), and males from ethanol or from living males and females.  The PCr product size (without the primers) of ITS2 sequences were 801 bp for Ps. comstocki, 773 bp for Pl. citri and 770 bp for Pl. ficus. The sequence variation among  Ps. comstocki males and females were between 0.375 – 0.0 and among Pl. citri between  0.26 and 0.00 (See Tab 3. and 4.). The amplified ITS2 sequences were compared with  the similar sequences in the Genebank. It is very interesting that Ps. comstocki collected  and characterized in Korea (1357 bp Genebank accession no. fj430147), and France (709  bp Genebank accession no.gu134668 and gu134669) showed very high identity with  species collected in Turkey (in case of gu134668 and hm628568 there is 100 %) The  same tendency could be observed in the case of Pl. citri and Pl. ficus. Pl. citri individuals  collected and characterized in France, Korea or Hungary showed 99.9 - 100 % identity.  ITS2 sequences of Pl. ficus individuals also showed 99.9 – 100 % identity regardless of  their origin, but differed from Pl. citri (90.4% identity). Pl. minor were also included in  the comparison test and showed 99.7 % identity with Pl. citri. Considerable divergence  was observed between the Ps. comstocki and Pl. citri (53.3 % – 56.1% identity), which  give possibility to identify and distinguish the males of these species (Fig. 1). The very low sequence variation of different individuals regardless their origin and  state (living, dry material or conserved in ethanol) indicate that ITS-2 molecular marker  is suitable method for reliable characterization and differentiation of Pl. citri and Ps. comstocki species. It was determined that males from the pheromone traps kept in 6-7  months in laboratory condition were suitable for molecular work. on basis of molecular  data it was possible to separate the males of Ps. comstocki and Pl. citri. The high diver- gence for the ITS 2 region suggest that it should be possible to design species-specific  primers and discriminate Ps. comstocki and Pl. citri males based on PCr method.  The molecular data could be a very important tool for the verification of the males  collected by pheromone traps, to exclude the accidentally present specimens in the trap,  and other species, for which the pheromone compound could serve as attractant. This  result is especially important in quarantine and ecological studies of distribution of  important pest species in new localities. aCKNoWLeDGeMeNTS The authors are grateful to the Hungarian Fund for research (oTKa) (No. 75889) for  financial support for this work, as well as to Dr. Panos Milonas and zsuzsanna Konczné  Benedicty for help in trapping works in Greece and in Hungary. 177I. Tóbiás et al.: Molecular tools for the identification of males of scale insects Ta b 3 - I de nt ity o f I TS 2 se qu en ce s o f P s. c om st oc ki c ol le ct ed fr om K or ea (f j4 30 14 7) , F ra nc e (g u1 34 66 8 an d gu 13 46 69 ) a nd T ur ke y (h m 62 85 68 – hm 62 85 71 ). (P s. co m -P C R 1 K oz ár F . r ef . n o. 9 25 4 an d Ps .c om -P C R 2 K oz ár F . r ef . n o. 9 25 4 ar e P C R p ro du ct s of s ep ar at e in di vi du al s) . gu 13 46 68 gu 13 46 69 hm 62 85 68 hm 62 85 69 hm 13 85 70 hm 13 85 71 P s. co m -P C R 1 P s. co m -P C R 2 fj 43 01 47 99 .3 0 99 .4 99 .2 99 .1 99 .1 99 99 .1 98 .9 gu 13 46 68   99 .9 10 0 99 .9 99 .7 99 .9 99 .9 99 .7 gu 13 46 69     99 .9 99 .7 99 .6 99 .7 99 .7 99 .6 hm 62 85 68       99 .8 99 .8 99 .9 99 .9 99 .7 hm 62 85 69         99 .6 99 .8 99 .7 99 .6 hm 13 85 70           99 .6 10 0 99 .6 hm 13 85 71             99 .6 10 0 P s. co m -P C r 1               99 .6 Ta b 4 - I de nt ity o f I TS 2 se qu en ce s of P l. ci tr i c ol le ct ed fr om K or ea (f j4 30 14 5) , F ra nc e (g u1 34 67 5 an d gu 13 46 78 ) a nd H un ga ry (h m 62 85 72 - hm 62 85 75 ), Pl . fi cu s an d Ps . c om st oc ki ( H M 62 85 69 ), (P l.c itr i- P C R 1 K oz ár F . r ef . n o. 9 20 3 an d Pl .c itr i- P C R 2 K oz ár F . r ef . n o. 9 46 2 ar e P C R p ro du ct s of s ep ar at e in di vi du al s) . gu 13 46 75 gu 13 46 78 hm 62 85 76 hm 62 85 75 hm 62 85 74 hm 62 85 73 hm 62 85 72 gu 13 46 76 gu 13 46 77 Pl .ci tri PC R1 Pl .ci tri PC R2 Pl .fic us t57 9-L 1/1 Pl .fic us T5 79 -L 3/1 HM 62 85 69 fj4 30 14 5 10 0 99 ,9 99 ,9 99 ,9 10 0 99 ,9 10 0 99 ,7 90 ,4 10 0 10 0 91 .4 91 .5 55 .6 gu 13 46 75   99 ,9 10 0 10 0 10 0 10 0 10 0 10 0 90 ,4 10 0 10 0 90 ,4 90 ,6 53 .3 gu 13 46 78     99 ,9 99 ,9 99 ,9 99 ,9 99 ,9 99 ,6 90 ,2 99 ,9 99 ,9 90 .3 90 .4 53 .5 hm 62 85 76       10 0 99 ,9 99 ,7 99 ,9 99 ,7 90 ,4 10 0 10 0 91 ,4 91 ,5 56 .0 hm 62 85 75         99 ,9 99 ,7 99 ,9 99 ,7 90 ,4 10 0 10 0 91 ,3 91 ,5 55 .9 hm 62 85 74           99 ,9 10 0 99 ,7 90 ,4 10 0 10 0 91 ,5 91 ,6 56 ,0 hm 62 85 73             99 ,9 99 ,7 90 ,4 10 0 10 0 91 ,4 91 ,5 56 ,0 hm 62 85 72               99 ,7 90 ,4 10 0 10 0 91 ,5 91 ,6 56 .1 gu 13 46 76                 90 ,4 99 ,7 99 ,7 90 ,4 90 ,5 49 .9 gu 13 46 77                   90 ,4 90 ,4 99 ,9 10 0 54 .3 Pl .ci tri -P Cr 1                     10 0 90 ,7 90 ,9 54 .3 Pl .ci tri -P Cr 2 90 ,8 90 ,9 54 .4 Pl . fi cu s t 57 9- L1 /1 99 ,9 57 .1 Pl . fi cu s t 57 9- L3 /1 57 .1 Journal of entomological and acarological research, Ser. II, 42 (3), 2010178 Fig. 1 - Comparison of ITS 2 sequences of Ps. comstocki and Pl. citri males. Percent Similarity: 55.906 Percent Identity: 55.906 Match display thresholds for the alignment(s): | = IDENTITY : = 5 . = 1 PcomstockiF.seg x PlanococcuscitriA5.seg January 12, 2011 09:56 . . . . . 1 TGCGGcCCTCGTG.ACCAAAGAGTCCTGGGCCACGCCTGTCTGAGGGTCG 49 ||||||||||| | || ||||||||||||||||||||||||||||||||| 1 TGCGGCCCTCGCGAaCAAAAGAGTCCTGGGCCACGCCTGTCTGAGGGTCG 50 . . . . . 50 GTCATACGTGTAACACGATGGTTGCGTTCTCGCGAGTTTCGCGCTCGCTC 99 || ||||||||||||||||||||||||||||||||| | ||| | 51 GTTATACGTGTAACACGATGGTTGCGTTCTCGCGAGCGCCTCGCAGTTTT 100 . . . . . 100 CGCAGCAGACCAGATCGTGCGCGCTCGTCGCGCGCGTGTGTGtgAATTCA 149 | | || | || | | | | ||| || | 101 CACCGCGAGGTTAGCTCGCCCCGTGACAGACCAGATCGAGCGTGTGTTTA 150 . . . . . 150 CGCACGCCGCCGAG...CGCGCGAGAGCGAGTACGTTGCGCTTTGCGGCG 196 ||||| | ||| |||| | ||| || | || | 151 CGCACATGCCAGAGCGACGCGGTTTTCGAATCGCGTCGCTCGAAGCTGAC 200 . . . . . 197 ACTTCGTACGCTCGAAGCTGACGATTCGTTTGCCCGCGTGACTTGTCGCG 246 |||| ||| | | | ||| | | ||| || || 201 GACTCGTTCGCGCTGACGCGGGCCATCGCCTCCGTGCGGTGGTTAGCGTC 250 . . . . . 247 TCGACGGCGAACGTTCGTCGAAATATTGCGGTTCTCGCCCGCATCGATAG 296 | || ||||||||||| |||||| | | ||| || 251 GTG.CGTCGAACGTTCGTTGAAATACACGGCGCCGATGCCGAGAAACAAG 299 . . . . . 297 TTCGTTCACGAACAACAACGTGTTCGACGAAAGCGTGCCGCGTGCAGCGG 346 |||||||||||||| | | ||| || | ||| | 300 TTCGTTCACGAACACCGTGTTCGAAAACGTTGCCGCGTATAGTGGTAGAG 349 . . . . . 347 ACCATATGCGAACGCGATGCGGTAGATACTCGGAGAGTGCGTTGGCCGTC 396 | | | ||| || || ||| || || | || 350 AGCGAAGGCGGTAGCTATCGTAGAGAGCGTCCGATGAACGGGCATCCAGA 399 . . . . . 397 GGATGTACGGGCGTCGAAAATTCTCTCTCGGGGCGACGGCGGGTGACGCG 446 | | | | ||| || | | | |||| 400 GAGAGGTGGTGGCGAGAAGAT..............ATACGGAGGATCGCG 435 . . . . . 179I. Tóbiás et al.: Molecular tools for the identification of males of scale insects 447 CGTGTTGTACGATCGCGCGTTCC.......AACACCCCCTCGTCGCTTGA 489 |||||||||||||||||||| | || | | ||| | 436 CGTGTTGTACGATCGCGCGTCTCCGAAAAAAAAAGCTTAACGTTACCGCC 485 . . . . . 490 GAAAAAAATTATACGACGCGAAGACGGTTTTCTCTTTACACGCCAACCGC 539 | || | |||||| ||||||||| | || || | || | 486 GTCGAATCTCATACGATGCGAAGACGTCTCTCGAAATAGAGGCGATAGTT 535 . . . . . 540 GATCGTTTCCCGATCGGACTTTGAGTATTTTTTCCGCAGTTCGCCCGCCT 589 |||||| | ||| | || || || || || | 536 TGCCGTTTCTCTATCCACGTACGA..........CGTTGTACGTGCGATT 575 . . . . . 590 GCACGACGACGTTGGCGCACGTACGCGCGCGCGTTAGCGTGTGTTCGCGT 639 || || |||| ||| || | || | | ||||| 576 GCGCG....CGTTcGcGTCGTTATTCCGGCCGTTGTCGACGAATTCGCTC 621 . . . . . 640 CGTCATTCCGGCCGTTGTcGACGAAATTCAATAATTCGAAGACGGCACTC 689 | | | || ||| || | | | || || 622 ACGCGTCATGTACGGACGCGATGATGACGATCGAGAACGGGCACACATTC 671 . . . . . 690 GGAAGGCGATTTCGCGACTCGCGTTACGTGCGACGCACGCGAACGTACCC 739 ||||| ||| | || | | | | ||| | ||| || | 672 GGAAGACGA.TGCGAGCGTGCGATAATTCGCGTTATAAGCGTACTT.... 716 . . . . . 740 GACACACACGCACGCATACGATTTCACGCCGACCTCAGATCAGGTAAGAC 789 | || | || | | ||||||||||||||||||||||||||| 717 ..CGTACCAGGCTTCACTCTGTATCACGCCGACCTCAGATCAGGtAAGAC 764 . 790 TACCCGCCGAAT 801 ||||||||| 765 TACCCGCCG... 773 Journal of entomological and acarological research, Ser. II, 42 (3), 2010180 reFereNCeS Ben-DoV y., MiLLer D. r., giBSon g. A. P., 2009 - ScaleNet: a database of the scale insects of  the World. Source: http//www.sel.barc.usda.gov/scalenet/scalenet.htm, using - Scales in a  region Query results.  Beuning L., MurPhy P., wu e., BAtCheLor t., MorriS B., 1999 - Molecular-based approach to  the differentiation of mealybug (Hemiptera: Pseudococcidae) species. -Journal of economic  entomology, 92, 463-472. DeVereux J., hAeBerLi P., SMithiS o., 1984 - a comprehensive set of sequence analysis program  for the VaX. - Nucleic acids. res., 12, 287-395. Frey J., Frey B., 1995 - Molecular identification of six species of scale insects (Quadraspidiotus  sp.) by raPD-PCr: assessing the field-specificity of pheromone traps. - Molecular ecology,  4, 777-780. goDinho M. A., FrAnCo J. C. 2001- Survey of the pest status of mealybugs in Portugese vineyards.  IoBC WPrS Bulletin, 24, 221-226. KiM h., Lee S., 2008 - Molecular systematics of the genus Megoura (Hemiptera: aphididae) using  mitochondrial and nuclear DNa sequences. - Molecules and Cells, 25: 510-522. KoSztArAB M., Kozár F., 1988 - Scale insects of Central europe - akadémiai Kiadó, Budapest,  456 pp. Kozár F., 1997 - Insects in a Changing World. - acta Phytopathologica et entomologica Hungarica,  32, 129-139. Kozár F., (ed.) 1998 - Catalogue of Palaearctic Coccoidea. - Plant Protection Institute, Hungarian  academy of Sciences, Budapest, 526 pp.  Kozár F., 2005 - Distribution records of scale insects in Hungary. - MTa Növényvédelmi  Kutatóintézete, Budapest, 136 pp. Kozár F., hiPPe C., MAni e., 1995 - Quadraspidiotus nembe tartozó pajzstetű fajok hímjeinek  morfológiai vizsgálata (Homoptera: Coccoidea) (in Hungarian) (Morphological study of  males of the Quadraspidiotus genus). In: Sáringer Gy., Seprős I. és Szemessy á. (szerk.). -  41. Növényvédelmi Tudományos Napok, Budapest. 51. Kozár F., hiPPe C., MAni e., 1996 - Morphometric analyses of the males of Quadraspidiotus  species (Hom., Diaspididae) found in european orchards or their vicinity. - Journal of applied  entomology, 120, 433-437. Kozár F., hiPPe C., MAni e., 1997 - Kulcs a Quadraspidiotus (Homoptera: Coccoidea) pajzstetű  nembe tartozó hímek meghatározásához (in Hungarian with english summary) (Key to de- termine Quadraspidiotus (Homoptera: Coccoidea) males and to distinguish them from males  of other genera). - Növényvédelem, 33, 321-327. Kozár F., nAgy DáViD A., 1986 - The unexpected northward migration of some species of insectes  in Central europe and the climatic changes. - anzeiger für Schadlingskunde, Planzenschutz  und Umweltschutz, 59, 90-94. Kozár F., SzentKiráLyi F., 2005 - Some effects of climate change on Insects in Hungary. In: Láng  I. (ed.) - Natural ecosystems, CD, 208-218. LAzAr J., KÖLBer M., FArKAS e., FArKAS g., toBiAS L., 2000 - Grapevine virus diseases and  clean grape stock program in Hungary. - 13th ICVG Conference, adelaide, 12-17 March 2000. MALAuSA t., FeniS A., wArot S., gerMAin J.F., riS. n., PrADo e., Botton M., VAnLerBerghe- MASutti F., SForzA r., CruAuD C., CouLoux A., Kreiter, P., 2010 - DNa markers to  disentangle complexes of cryptic taxa in mealybugs (Hemiptera: Pseudococcidae) - Journal  of applied entomology, 135(1-2):142-155.  181I. Tóbiás et al.: Molecular tools for the identification of males of scale insects PArK D-S., LeeM y.J., hAhn K.w., Suh S-J., hong K-J., oh h-w., 2010 - Molecular identification  of mealybugs (Hemiptera: Pseudococcidae) found on Korean Pears. - Journal of economic  entomology, 103, 25-33. PeLLizzAri SCALtriti g., 1991 - recent data on Italian fauna of Homoptera Coccoidea. atti XVI  Congresso nazionale italiano di entomologia, Bari - Martina Franca (Ta) 23/28 settembre  1991, 763-769.  rung A., SCheFFer S. J., eVAnS g., MiLLer D., 2008 - Molecular identification of two closely  related species of mealybugs of the genus Planococcus (Homoptera: Pseudococcidae). -  annals of the entomological Society of america, 101, 525-532. SAMBrooK J., FritSCh e.F., MAniAtiS t.A., 1989 - Molecular cloning: a laboratory manual. - Cold  Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1659 pp. SentenAC g., KuntzMAnn P., 2003 - etude des Cochenilles et des antagonistes qui leur sont  associés dans des vignobles en Bourgogne et en alsace de 2000 à 2002. - IoBC WPrS  Bulletin, 26, 247-252. SForzA r., BouDon-PADieu e., greiF C. 2003 - New mealybug species vectoring grapevine  leafroll associated viruses 1 and 3 (GLraV-1 and 3). - european Journal of Plant Pathology,  109, 975-981. iStVán tóBiáS - Plant Protection Institute, Hungarian academy of Sciences, H-1525, P.o.Box  102, Budapest, Hungary. tobias@julia-nki.hu FerenC Kozár - Plant Protection Institute, Hungarian academy of Sciences, H-1525, P.o.Box  102, Budapest, Hungary. BorA. M. KAyDAn - Yüzüncü Yıl Üniversity, agriculture Faculty, Plant Protection Department,  65080 Campus, VaN. KingA FetyKó - Plant Protection Institute, Hungarian academy of Sciences, H-1525, P.o.Box  102, Budapest, Hungary. accepted 20 December 2010