Impaginato 14 Adv. Hort. Sci., 2011 25(1): 14-20 Received for publication 15 September 2010. Accepted for publication 14 January 2011. Radiation-induced chromosomal aberrations in grape phylloxera H. Makee, N. Tafesh, I. Idris Department of Biotechnology, Atomic Energy Commission of Syria, PO Box 6091, Damascus, Syria. Key words: chromosome aberrations, grape phylloxera, irradiation, reproduction. Abstract: Chromosomal aberrations in phylloxera females induced by different doses of gamma irradiation were detected. The results showed that the chromosomes of all tested embryos of irradiated phylloxera had aber- rations, regardless of dose. When phylloxera nymphs were irradiated, the chromosomal number on the metaphase plate of some embryos’ cells was increased. The result indicated that the chromosomal aberrations influenced the mortality, longevity and reproduction of phylloxera. Eight autosomal chromosomes were identi- fied according to their length. Additionally, the karyotype of irradiated and unirradiated populations of local phylloxera strain was defined. 1. Introduction Grape phylloxera, Daktulosphaira vitifoliae Fitch (Homoptera: Phylloxeridae), an aphid-like gall-form- ing parasite, is one of the most destructive insect pests of cultivated grape Vitis vinifera L. world wide. Grape phylloxera causes direct damage to grapevine by form- ing damaging root galls. These galls are metabolically active organs suited to match the nutritional require- ments of phylloxera and can support populations with high reproductive rates. Granett et al. (1985) reported that there was frequent decline of commercial vine- yards as result of this pest, and consequently losses of quality and yield of grapes. The use of resistant rootstocks is the most common and effective means of managing phylloxera. Our pre- vious studies showed that some rootstocks were more resistance than others to grape phylloxera (Makee et al., 2003). However, for unknown reasons, the resis- tance of some rootstocks may break down and farmers must replant vineyards (Granett et al., 1983; Song and Granett, 1990; De Benedictis and Granett, 1993). Therefore, additional ways to control this pest should be considered. Sanitation and quarantine can be considered as required procedures to prevent the movement of this serious pest. Insecticides and hot water dip treatments are used as quarantine treatments (Granett et al., 2001). Ionizing radiation has been recognized as an alternative method for treating agricultural products to overcome quarantine barriers in trade (FAO, 2003). Irradiation treatment does not influence the quality of many com- modities; it is reasonably safe to the consumers and environment. Previously, several authors have present- ed reviews of this subject (Hallman, 1998; Johnson and Marcotte, 1999; Hallman, 2000, 2001). Irradiation has been successfully used for the con- trol of many insect pests such as codling moth Cydia pomonella (L.), beetle Prionolplus reticularis White (Lester et al., 2000), apple maggot, Rhagoletis pomonella (Walsh), the borer Eucosma notanthes Meyrick (Lin et al., 2003), coconut scale Aspidiotus destructor Signoret (Follett, 2006), the weevil Ster- nochetus mangiferae (F.) (Follett, 2001), cigarette bee- tle, Lasioderma serricorne (F.) (Hu et al., 2002), and the rice weevil Sitophilus zeamais Motschulsky (Hu et al., 2003). Makee et al. (2008) proposed that gamma irradia- tion could be economically very useful in quarantine treatments against phylloxera. The results showed that the percentage of matured phylloxera females and fecundity were markedly reduced when higher doses of gamma irradiation were used (Makee et al., 2008). However, to our knowledge efforts of associate perfor- mance parameters of irradiated phylloxera with chro- mosomal rearrangements have not yet been studied. Therefore, the purpose of the present research was to detect chromosomal aberrations in phylloxera females induced by different doses of gamma irradiation. The influence of such chromosomal aberrations on longevi- ty and reproduction of phylloxera was examined. Moreover, determination of the number and length of autosomal and sex chromosomes was undertaken. Such 15 study will allow definition of the karyotypes of irradi- ated and unirradiated populations of the local phyllox- era strain. 2. Materials and Methods Establishment of phylloxera colony Grape phylloxera was originally collected from field-infested roots of the local grape varieties in south- ern parts of Syria. All insects were reared on fresh and healthy pieces of local grape variety Balady roots, 4-7 mm in diameter and 5-7 cm long as outlined in Makee et al. (2003). The experiments were conducted at 25±1°C with 70±5% RH, and 24 hr darkness. Egg ster- ilization was carried out as described by Makee et al. (2003). A Co60 source (Issledov Gamma Irradiator, Techsnabexport Co. Ltd., Moscow, Russia) delivering a dose rate of 60 Gy/min was used to treat the insects. Effect of irradiation on matured females, fecundity, and oviposition period New phylloxera eggs were placed on fresh root pieces and left until hatching. A group of three-week- old feeding phylloxera nymphs was taken. Nymphs were irradiated at different doses: 0-10-20 and 30 Gy (n= 25 nymphs at each dose). Irradiated and unirradiat- ed nymphs were kept at 25±1°C with 70±5% RH, and 24 hr darkness. A daily microscopic inspection of all phylloxera stages at each applied dose was carried out. The num- ber of feeding nymphs, which were able to develop to adult stage, was observed to determine the percentage mortality at each dose. Female fecundity and longevity was determined at each tested dose. Chromosome preparations Mitotic metaphase chromosomes were obtained from 24 to 36-hr-old embryos. At each dose, 10 eggs were taken and placed in a 1.5 ml tube. The eggs in each tube were fixed in Carnoy’s fixative (ethanol:chloro- form:acetic acid 6:3:1) and shaken for 10 min. Then a drop was taken and transferred onto a clean slide. Short- ly before drying, a drop of 60% acetic acid was added and macerated for 2-3 min with fine tungsten needles. Then the specimen was spread on the slide using a heat- ing plate at 45°C to allow evaporation of the acetic acid. The preparation was then stained and mounted in lactic acetic orcein for 5 min; redundant stain was removed with a piece of filter paper. The cover glass was sealed with nail polish. Chromosome preparations were exam- ined in phase contrast micrographs. Chromosome measurement The lengths of the chromosomes from 12 well spread orcein-stained metaphase chromosomes were measured in digital images using Digitizier software, version 1 (developed by the Mathematics Department, Atomic Energy Commission of Syria). Chromosome lengths were ranked for each cell nucleus and means and standard deviations (SD) were calculated. Relative chromosome lengths were calculated as percentages of the total length of all chromosomes in the diploid set. 3. Results Effect of irradiation on mortality, longevity and fecun- dity Our results show that the percentage mortality of irradiated phylloxera nymphs was significantly higher than that of unirradiated ones. A regression line was fit- ted to present the relationship between gamma irradia- tion and percentage mortality of phylloxera (Fig. 1), showing that the percentage mortality was positively correlated with dose (t= 7; P=0.05). The lowest per- centages of mortality were recorded at 10 Gy, after which the percentages started to increase. Only about 16% of phylloxera nymphs were able to reach matured female stage at 30 Gy. % m or ta lit y Fig. 1 - Effect of gamma irradiation on percentage mortality of grape phylloxera. Table 1 - Effect of different doses of gamma irradiation on mean num- ber of eggs and longevity of phylloxera Dose (Gy) Mean no. eggs(±SE) Mean longevity (d) (±SE) 0 10 20 30 60.0±4.0 a 24.5±2.7 b 10.7±1.0 c 0.7±0.2 d 14.6±0.75 a 10.0±0.88 b 7.0±0.69 c 0.6±0.01 d Means followed by different letters (columns) are significantly differ- ent at P< 0.05 (Tukey HSD test). Table 1 illustrates that phylloxera longevity and number of eggs were considerably influenced by the applied dose (Table 1). The mean value for longevity and the mean number of eggs were significantly reduced by increasing the dose (F=75.67; df=3, 96; P=0.05 and F=106.78; df=3, 96; P=0.05, respectively). 16 Chromosomal analysis under a light microscope From matured phylloxera females, 24 to 36-hr-old embryos, which contain a higher proportion of dividing cells, were taken to analyze the metaphase chromo- somes. It was revealed that the wild-type metaphase karyotype of phylloxera consists of 10 chromosomes. There were eight autosomal chromosomes and sex chromosomes (XX) (Fig. 2 A). A karyotype of 2n=9 was also found (Fig. 2 B). Based on our observations, phylloxera metaphase chromosomes appeared like thick condensed rods. In some cells, an additional very small chromosome was detected (Fig. 3). A B 1 µm 1 µm Fig. 2 - Normal metaphase kryotype from embryonic cells of Grape phylloxera, Daktulosphaira vitifoliae Fitch: A) 2n= 10; B) 2n=9 + small chromosome (the arrow). 1 µm Ý Fig. 3 - Normal metaphase kryotype from embryonic cells of Grape phylloxera: 2n= 10 + small chromosome (the arrow). To study the effect of different doses of gamma irra- diation on phylloxera chromosomes, 24 to 36-hr-old embryos were examined at each tested dose, and it was found that the chromosomes of all tested embryos of irradiated phylloxera had aberrations, regardless of dose. At all different doses, sticky chromosomes were observed in the embryo cells (Fig. 4). When 20 Gy was applied, inter-chromosome translocations were clearly visible (Fig. 5). However, increasing in the chromoso- mal number on the metaphase plate in some cells was noticed in embryos when phylloxera nymphs were irra- diated (Fig. 6). A B 1 µm 1 µm C D 1 µm 1 µm E 1 µm Fig. 4 - Mitotic metaphase chromosomes from embryos of treated Grape phylloxera with gamma ray, showing sticky chromo- somes at deferent doses: A-B at dose 10 Gy; C) at dose 20 Gy, D-E) at dose 30 Gy. Fig. 5 - Orcein-stained preparations from embryos of irradiated phyl- loxera at dose 20 Gy showing mitotic metaphase chromo- somes with a translocation on the large chromosome (the arrow). Ý 1 µm 17 Chromosomes measurement It was possible to identify eight autosomal chromo- somes according to their length when different metaphase chromosomes were investigated. Table 2 illustrates the mean length and relative length for each identified chromosome. The total mean length of com- plete metaphase chromosomes was 12.17±1.97 µm. Chromosome pair no. 1 is an extra large chromo- some with an average total length of 2.31±0.3 µm and relative length of 18.18%; chromosome pair no. 2 is a large chromosome with an average total length of 1.33±0.3 µm and relative length of 10.5%; chromo- some pair no. 3 is a medium chromosome (1.12±0.65 µm); and chromosome pair no. 4 is a short chromo- some (0.90±0.160 µm) (Table 2). The sex chromosome (XX) was clearly recognized. The sex chromosome is the shortest chromosome in the metaphase complements with an average total length of 0.69±0.1 µm and relative length of 5.51%. The mean length of the additional chromosome, which was observed in several cells, was only 0.5±0.1 µm. considerable variation was noted among the species tested within these orders (Makee and Saour, 1999; Bakri et al., 2005; Follett et al., 2007). In fact, a few studies were carried out to determine the radioresis- tance of Hemiptera (scales, mealy bugs, aphids, and whiteflies). A previous study showed that the egg hatch of phyl- loxera decreased when eggs were subjected to high doses of gamma irradiation and the percentage of matured phylloxera females significantly increased as older nymphs and lower doses were used (Makee et al., 2008). On the contrary, fecundity was markedly reduced when older nymphs and higher doses were employed. However, a relationship between chromoso- mal aberrations induced by irradiation and phylloxera biology was not determined in the study. Phylloxera can be considered a cytogenetically exciting insect species because of abnormal features related to its cyclical parthenogenesis, and because it has holocentric chromosomes (chromosomes that lack a localized centromere). Phylloxera populations can consist totally of parthenogenetic (thelytokous) females. Several studies showed that grape phylloxera mainly reproduces asexually (parthenogenesis): an egg cell can develop into offspring without fertilization by a sperm. Thus, the offspring and its siblings are assumed to be genetically identical to the mother (Vor- werk and Forneck, 2006). Parthenogenetic reproduc- tion of phylloxera has been observed in the field and can be easily maintained under constant conditions in the greenhouse or in vitro. This type of reproduction allows phylloxera populations to be replicated several fold, thus several asexual generations can be analyzed within a short period. Once a year, XX parthenogenet- ic phylloxera females, like aphids, produce one egg that develops as an XO male, having lost half its X chromatin during the single maturation division (Blackman and Hales, 1986; Blackman, 1987). The current study has shown that phylloxera nymph mortality increased with irradiation (Fig. 1), confirm- ing the results reported by Makee et al. (2008). Corre- spondingly, Dohino et al. (1998) found that the sur- vival of aphids was significantly decreased when they were treated with doses of 400-600 Gy. The high death rate, especially when phylloxera nymphs were exposed to higher doses of gamma irradiation, can be attributed to the effects of the dominant lethal mutations induced in phylloxera nymphs’ chromosomes by irradiation (LaChance, 1967). When low doses were applied, a small portion of irradiated nymphs successfully com- pleted development and produced matured females, survival which was due to the holokinetic nature of phylloxera chromosomes. Irradiation can cause frag- mentation but the resulting fragments are still able to move on the mitotic spindle so that chromosome break- age does not lead automatically to the loss of genetic material (Hughes-Schrad and Ris, 1941). Our results reveal that the fecundity and longevity Fig 6 - Orcein-stained preparations from embryos of irradiated phyl- loxera showing: A) at dose 20 Gy: a cell with about 15 chro- mosomes which some of them are stuck together; B) at dose 30 Gy: a cell with 14 chromosomes. A B 1 µm 1 µm Table 2 - The mean length and relative length (%) of phylloxera chro- mosomes No. chromosome Mean length (µm±SD) Relative length (%) 1 1 2 2 3 3 4 4 X X Total mean length 2.31±0.3 2.31±0.3 1.33±0.3 1.33±0.3 1.12±0.65 1.12±0.66 0.90±0.16 0.90±0.16 0.69±0.1 0.69±0.1 12.17±1.97 18.18 18.18 10.5 10.5 8.81 8.81 7.1 7.1 5.41 5.41 100 4. Discussion and Conclusions Several studies demonstrated that Dipteran, Coleopteran and Hemipteran species tend to be more radiosensitive than Lepidopteran species. However, 18 of surviving matured females, irradiated as nymphs, were greatly impacted by irradiation (Table 1). Com- parable results were reported when crawls and nymphs of mealybug, Maconellicoccus hirsutus (Green), were irradiated (Jacobsen and Hara, 2003). Therefore, at low doses some matured phylloxera females were recorded, but they laid only few eggs and lived for a short period of time. It could be that the induced chromosomal aber- rations in irradiated nymphs prevent the normal process of mitotic division, which leads to egg produc- tion. Therefore, the matured females were unable to produce a normal number of eggs. To study the effect of different doses of gamma irra- diation on phylloxera chromosomes, 24 to 36-hr-old embryos were examined at each tested dose. It was noted that the chromosomes of all tested embryos of irradiated phylloxera had aberrations, regardless of dose. We noticed sticky chromosomes, inter-chromo- some translocations, and increases in the chromosomal number on the metaphase plate in some cells. All these chromosomal aberrations in the embryos were expect- ed as the phylloxera, like Lepidptera species, has holo- centric chromosomes. It is reported that irradiation causes fragmentations and translocations in many species of Lepidptera (Traut et al., 1986; Makee and Tafesh, 2006, 2007). And because the chromosomes are holocentric when a break occurs, the fragments are usually not lost and can still be attached on the spindle. In the present study on phylloxera, a lot of chromoso- mal breaks occurred during the formation of eggs in the ova of the nymphs which gave sticky chromosomes and inter-chromosome translocations in the cells of the embryos. It can also be said that spindles were affected by the irradiation which caused an increase in the chro- mosomal number on the metaphase plate in some embryo cells. In this work, the embryos of laid eggs varied great- ly in their chromosomal rearrangements (Figs. 2, 3, 4 and 5). However, such rearrangements allow the for- mation of embryos but it is unknown if they will per- mit the development of embryos until egg hatch. In mealybug only embryos with an approximately normal amount of paternal chromosomal material were able to survive (Nelson-Rees,1962). The current study has shown that the metaphase complement of the Syrian strain of the phylloxera female consists of 10 chromosomes, representing eight autosomal and two sex chromosomes, confirming the results presented by Forneck et al. (1999) and Maillet (1957). We found also normal karyotype with 2n= 9 in the embryonic cells coincidently. Forneck et al. (1999) found one karyotype containing 2n = 9 in the somatic cells of phylloxera and they interpreted it as a male sexual phylloxera, although they did not find any sper- matides during their study, which leads us to think that maybe this deficiency in chromosome number is a kind of variety of the karyotype. When defining the karyotype of six phylloxera pop- ulations from Germany, Forneck et al. (1999) noticed extra chromosomes. Similarly, in our study an addi- tional very small chromosome was observed in some examined cells of the Syrian phylloxera strain. The detection of supernumerary chromosomes was reported in aphids as well (Blackman, 1976; Wilson et al., 2003). Such supernumerary or accessory chromosomes are not essential for the life of a species and are lacking in most of the individuals; they do not carry genes nec- essary for basic growth, but may have some functional significance such as to increase asymmetry chiasma distribution or increase variation by increasing crossing over and recombination frequencies. In aphid, Blackman (1980, 1981) suggested that the differences in chromosome numbers might be due to dissociations or fusions involving elements of the nor- mal diploid set, or to the presence of supernumerary B chromosomes. The centromeric activity of holocentric chromosomes, dispersed along its full length, allows the broken chromosomal fragments to segregate during mitosis (Ris, 1942). Moreover, Blackman (1980) pro- posed that thelytokous reproduction of aphids is a fac- tor that permits karyotype variation within populations of the same species. The phylloxera karyotype consists of 10 chromo- somes. The total complement length is about 12Ìm and the chromosomes range in length from 0.7 to 2 Ìm. When mitosis of Agallia constricta (leafhopper) was examined, the metaphase chromatin appeared to be a 2-3 Ìm wide (Rieder et al., 1990). Based on embryo metaphase, the chromosomes of phylloxera females could be sorted into five different size-dependent groups: extra long, long, medium, short and extra short (Table 2). However, in some cells a dot-like chromo- some, that represents the additional chromosome, was observed. Forneck et al. (1999) classified phylloxera chromosomes into two classes: one pair of large chro- mosomes and four pairs of shorter chromosomes. Nev- ertheless, in their study they did not mention the exact length of each pair. The X chromosome is the shortest one in phylloxera karyotype Forneck et al. (1999). However, Blackman et al. (2003) reported that in most aphids species the X chromosomes could be identified as the longest or sec- ond longest pair. On the contrary, in some aphids the X chromosome was the shortest pair (Blackman, 1986; Blackman et al., 2003). The present study confirms the efficiency of cyto- genetic techniques in analyzing the karyotype and chromosomal length of phylloxera, as well as tracing chromosome aberrations in irradiated phylloxera popu- lations. This investigation is a contribution to the search for genetic variation of phylloxera behaviour and development from different populations and pro- vides useful information that can be taken into account in pest management and quarantine measurements against phylloxera. However to apply irradiation tech- nology more comprehensive studies are still needed. 19 References BAKRI A., HEATHER N., HENDRICHS J., FERRIS I., 2005 - 50 Years of radiation biology in entomology: lessons learned from IDIDAS. - Ann. Entomol. Soc. Am., 98: 1-12. BLACKMAN R.L., 1976 - Cytogenetics of two species of Eur- eraphis (Homoptera, Aphididae). Chromosoma, 56: 393-408. BLACKMAN R.L., 1980 - Chromosome numbers in the Aphididae and their taxonomic significance. - Syst. Entomol. 5: 7-25. BLACKMAN R.L., 1981- Species, sex and parthenogenesis in aphids, pp. 75-85. - In: FOREY P.L. (ed.) The evolving bios- phere. Cambridge University Press, London, UK. BLACKMAN R.L., 1986 - The chromosomes of Japanese Aphidi- dae (Homoptera), with notes on the cytological work of Orihay Shinji. - Cytologia, 51: 59-83. BLACKMAN R.L., 1987 - Reproduction, cytogenetics and devel- opment, pp. 163-195. - In: MINKS A.K., and P. HARREWIJN (eds.) Aphids, their biology, natural enemies and control, Vol. 2A. Elsevier Science Publishers, Amsterdam, The Netherlands. BLACKMAN R.L., BROWN P.A., RAMIREZ C.C., NIEMEYER H.M., 2003 - Karyotype variation in the South American aphid genus Neuquenaphis (Hemiptera, Aphididae, Neuquenaphidi- nae). - Heredity, 138: 6-10. BLACKMAN R.L., HALES D.H., 1986 - Behaviour of the X chro- mosomes during growth and maturation of parthenogenetic eggs of Amphorophora tuberculata (Homoptera, Aphididae), in relation to sex determination. - Chromosoma, 94: 59-64. DE BENEDICTIS J., GRANETT J., 1993 - Laboratory evaluation of grape roots as host of California grape phylloxera biotypes. - Am. J. Enol. Vitic., 44(3): 285-291. DOHINO T., MATSUOKA I., TAKANO T., HAYASHI T., 1998 - Effects of electron beam irradiation on Myzus persicae (SULZ- ER) (Homoptera: Aphididae). - Research Bulletin of the Plant Protection Service, 34: 15-22. FAO, 2003 - Guidelines for the use of irradiation as a phytosani- tary measure. International Plant Protection Convention, ISPM no. 18. - Food and Agricultural Organization (FAO), Rome, Italy. FOLLETT P.A., 2001 - Irradiation as a quarantine treatment for mango seed weevil (Coleoptera: Curculionidae). - Proc. Hawaiian Entomol. Soc., 35: 95-100. FOLLETT P.A., 2006 - Irradiation as a phytosanitary treatment for Aspidiotus destructor (Homoptera: Diaspididae). - J. Econ. Entomol., 99: 1138-1142. FOLLETT P.A., YANG M.M., LU K.H., CHEN T.W., 2007 - Irra- diation for postharvest control of quarantine insects. - For- mosan. Entomol., 27: 1-15. FORNECK A., JIN Y., WALKER A., BLAICH R., 1999 - Kary- otype studies on grape phylloxera (Daktulosphaira vitifoliae Fitch). - Vitis, 38(3): 123-125. GRANETT J., BISABRI-ERSHADI B., CAREY J., 1983 - Life tables of phylloxera on resistant and susceptible grape root- stocks. - Ent. Exp. & Appl., 34: 13-19. GRANETT J., TIMPER P., LIDER L.A., 1985 - Grape phylloxera (Daktulosphaira vitifoliae) (Homoptera: Phylloxeridae) bio- types in California. - Journal of Economic Entomology, 78: 1463-1467. GRANETT J., WALKER M.A., KOCSIS L., OMER D.A., 2001- Biology and management of grape phylloxera. - Annu. Rev. Entomol., 46: 387-412. HALLMAN G., 1998 - Ionizing radiation quarantine treatments. - An. Soc. Entomol. Brasil, 27: 313-323. HALLMAN G., 2000 - Expanding radiation quarantine treat- ments beyond fruit flies. - Agric. for Entomol., 2: 85-95. HALLMAN G., 2001 - Irradiation as a quarantine treatment. - In: MOLINS R.A. (ed.) Food irradiation: principles and applica- tions. Wiley, New York, USA. HU T., CHEN C., PENG W.K., 2002 - The lethal effect of gamma radiation on Lasioderma serricorne (Fabricius) (Coleoptera: Anobiidae). - Formos. Entomol., 22: 157-162. HU T., CHEN C., PENG W.K., 2003 - Lethal effect of gamma radi- ation on Sitophilus zeamais (L.) (Coleoptera: Curculionidae). - Formos. Entomol., 23: 145-150. HUGHES-SCHRADER S., RIS H., 1941 - The diffuse spindle attachment of coccids, verified by the mitotic behavior of induced chromosome fragments. - J. Exptl. Zool., 87: 429-456. JACOBSEN C.M., HARA A.H., 2003 - Irradiation of Maconellic- occus hirsutus (homoptera: Pseudococcidae) for phylosanita- tion of agricultural commodities. - J. Econ. Entomol., 96(4): 1334-1339. JOHNSON J., MARCOTTE M., 1999 - Irradiation control of insect pests of dried fruits and walnuts. - Food Technol., 53: 46-48. LACHANCE L.E., 1967 - The induction of dominant lethal muta- tions in insects by ionizing radiation and chemicals as related to the sterile male techniques of insect control. - In: WRIGHT J., and R. PAL (eds.) Genetics of insects vectors of disease. Elsevier Science Publisher, Amsterdam, The Netherlands, pp. 813. LESTER P.B., ROGERS D.J., PETRY R.J., CONOLLY P.G., ROBERTS P.B., 2000 - The lethal effects of gamma irradiation on larvae of the huhu beetle, Prionoplus reticularis: a potential quarantine treatment for New Zealand export pine trees. - Entomol. Exp. Appl., 94: 237-242. LIN J.Y., HORNG S.B., HUNG C.C., 2003 - Effects of gamma radiation on survival and reproduction of the carambola fruit borer, Eucosma notanthes Meyrick (Lepidoptera: Tortricidae). - Formos. Entomol., 23: 189-197. MAILLET P., 1957 - Sur les chromosomes de quelques phyllox- erides de France. - Vitis, 1: 153-155. MAKEE H., CHARBAJI T., AYYOUBI Z., IDRIS I., 2003 - Eval- uating resistance of some rootstocks to grape phylloxera with an in vitro and excised root testing systems. - In vitro Cell. Dev. Biol. - Plant, 40(2): 225-229. MAKEE H., SAOUR G., 1999 - Non-Recovery of fertility in par- tially sterile male Phthorimaea operculella (Lepidoptera: Gelechiidae). - J. Econ. Entomol., 92(3): 516-520. MAKEE H., TAFESH N., 2006 - Sex chromatin body as a marker of radiation-induced sex chromosome aberrations in the pota- to tuber moth, Phthorimaea operculella (Lepidoptera: Gelechi- idae). - J. Pest Sci., 79: 75-82. MAKEE H., TAFESH N., 2007 - Sex chromatin body as a cytoge- netic marker of W chromosome aberrations in Cydia pomonel- la females. - In: VREYSEN M.J.B., A.S. ROBINSON, and J. HENDRICHS (eds.) Area-wide control of insect pests. From research to field implementation. Springer, Dordrecht, The Netherlands, pp. 792. MAKEE H., TAFESH N., MAREC F., 2008 - Analysis of radia- tion-induced W chromosome aberrations in codling moth Cydia pomonella (L.) by fluorescence in situ hybridization techniques. - J. of Pest Science, 81: 143-151. NELSON-REES W.A., 1962 - The effects of radiation damaged heterochromatic chromosomes on male fertility in the Mealy Bug, Planococcus Citri (Risso). - Genetics, 47(6): 661-683. RIEDER C.L., BOWSER S.S., COLE R., RUPP G., PETERSON A., ALEXANDER S.P., 1990 - Diffuse kinetochores and holo- kinetic anaphase chromatin movement during mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20. - Cell Motility and the Cytoskeleton, 15: 245-259. RIS H., 1942 - A cytological and experimental analysis of the mei- otic behavior of the univalent X chromosome in the bearberry aphid Tamalia (= Phyllaphis) coweni (Ckll.). - J. Exp. Zool., 90: 267-330. SONG G.C., GRANETT J., 1990 - Grape phylloxera (Homoptera: Phylloxeridae) biotypes in France. - J. Econ. Entomol., 83: 489-493. TRAUT W., WEITH A., TRAUT G., 1986 - Structural mutants of the W chromosome in Ephestia (Insecta, Lepidoptera). - Genet- 20 ica, 70: 69-79. VORWERK S., FORNECK A., 2006 - Reproduction mode of grape phylloxera (Daktulosphaira vitifoliae, Homoptera: Phyl- loxeridae) in Europe: molecular evidence for predominantly asexual populations and a lack of gene flow between them. - Genome, 49: 678-687. VORWERK S., SONNTAG K., BLAICH R., FORNECK A., 2008 - Application of current in situ hybridization techniques for grape phylloxera (Daktulosphaira vitifoliae, Fitch) and grape- vine (Vitis spp. L.). - Vitis, 47(2): 113-116. WILSON A.C.C., SUNNUCKS P., HALES D.F., 2003 - Heritable genetic variation and potential for adaptive evolution in asexu- al aphids (Aphidoidea). - Biological Journal of the Linnean Society, 79: 115-135.