Nuclear Polyhedrosis Virus Replication in P e r m a n e n t Cell Lines o f the C a b b a g e M o t h (Mamestra brassicae L.) H.G. Miltenburger and P. David Zoologisches institut der Technischen Hochschule Darmstadt
Fig. 1. (a) Lage und Form der Kristallnadeln innerhalb der Oehfmsewand werden durch die organische Hiillsubstanz markiert. Der Calcit wurde w/ihrend der Fixierung herausgel6st. Die Kristalle sind zufallsgem/iB orientiert, eine Matrix zwischen ihnen fehlt. (b) Nur w/ihrend der Geh/iusebildung finden sich im Cytoplasma Vesikel, die jeweils mehrere Kristallnadeln mit ihren Hfillen enthalten. Pr~iparation wie bei (a)
Elektronenmikroskopische Untersuchungen an Calzituba polymorpha Roboz haben nun gezeigt, dab hier die Kristallnadeln der Gehfiusewand in cytoplasmatischen Vesikeln gebildet und erst dann nach augen transportiert und zu einer Wand zusammengeftigt werden (Fig. 1 b). Ffir die Kristallanordnung innerhalb der Wandung ist also nicht eine epitaktische Anlage von Kristallkeimen auf oder in einer Matrix verantwortlich, sondern Transport-Pseudopodien, welche die vorgefertigten KristaUnadeln auf einer organischen Membran weitgehend zufallig anlagern. Calcitkristalle miissen gegeniiber dem umgebenden Medium vor Aufl6sung geschiitzt werden. Bei den bisher untersuchten Foraminiferen ist das Geh/iuse allseits yon einer organischen Schicht fiberzogen. Bei Calzituba I/igt sich eine/iul3ere Htillschicht jedoch nicht nachweisen. Darer ist jede einzelne Kristallnadel in einer organischen Hfille verpackt (Fig. 1). Erstmals zeigen sich hier auch Parallelen in den Morphogeneseablfiufen zwischen einer Foraminifere und den Testaceen, deren Geh/iuse ebenfalls aus im Cytoplasma vorgefertigten Bauelementen (Protein oder Calcit) erstellt wird [3]. Ich danke der Deutschen Forschungsgemeinschaft ffir die finanzielle Unterstfitzung dieser Arbeit (SFB 53). Eingegangen am 27. Januar 1976" 1. Towe, K.M., Cifelli, R.: J. Paleontol. 41, 742 (1967) 2. Haake, F.-W.: J. Foraminiferal Res. 1, 187 (1971) 3. Netzel, H. : Z. Zellforsch. 135, 45 (1972) Naturwissenschaften 63 (1976)
9 by Springer-Verlag 1976
The in vitro replication of nuclear polyhedrosis viruses (NPV) in monolayer cultures of insect cell lines has already been described [1]. This kind of investigation may render new knowledge with regard to the possibility of biological pest control with species-specific viruses since, for example, NPV are responsible for diseases in various lepidopterous larvae like the cotton bollworm (Heliothis zea). Therefore further experiments on in vitro NPV replication are of increasing interest. Five cell lines of the cabbage moth (Mamestra brassicae) were derived from tissue of four/five instar larvae in our laboratory 14-18 months ago: Mb 1203 (mixed explant from the ovary and the dorsal vessel), Mb 0503, Mb 2006, Mb 2007, and Mb 2506 (hemocytes). The cells have so far been subcultured more than a hundred times in a modified Grace-medium [2] and supplemented with 5 % fetal calf serum at 28 ~ in polystyrene plastic flasks (Falcon Plastics; Oxnard, California) or 180-ml glass bottles (Schott, Mainz). The doubling time ranges from 14-36 h depending on the cell line. In the monolayer cultures, most of the predominantly spindlelike cells adhere firmly to the vessel surface. For several months Mb 0503 and Mb 1203 have been growing very well in suspension cultures with a doubling time of 24-26 h. The morphology, culture-, and colony-growth curves as well as chromosome numbers per cell were used as parameters for the regular control of cell-line characteristics. As reported earlier, in somatic lepidopteran tissues, the cells may be highly polyploid or mixoploid with chromosome numbers up to 128 n [3]. Analyses of chromosome preparations from our cell lines showed in several hundred cells mixoploidy. Most cells have many more chromosomes than in the diploid set: 150 250, sometimes more than 300. The chromosomes are, with few exceptions, of minute size but all have obviously diffuse centromeres. We were able to determine the haploid number of meiotic stages of larval testis cells as 1 n = 31. To evaluate if our Mb cell lines would be suitable for NPV replication we infected two of them (Mb 0503, Mb 1203) that proliferated best, with infectious hemolymph of Autographa californica (Alfalfa looper), the NPV of which is known to have a relatively wide host-spectrum specificity [4]. Twelve h after introducing 0.05 ml of a cell- and inclusion body-free supernatant (from hemolymph of heavily infected larvae) into young cultures in both Mb-lines 80 to 90% of the early logphase cells showed cytopathic effects: nuclear granulation and hypertrophy. Two days later in each of these cells up to 100 cubic polyhedra had developed (Fig. 1 a). The centrifuged cell- and polyhedra-free supernatant from these cultures was again infectious to the same extent when introduced into healthy monolayer Mb cultures. We then infected the cell lines with infectious supernatant of centrifuged M. brassicae larval hemolymph. Again after 6-12 h cytopathic effects appeared but to a lessser degree. Ten to 30% of the cells showed alteration of the nuclei and developed up to 30 polyhedral inclusion bodies within the following six days (Fig. 1b). Although the supernatant of these cultures again induces cytopathic effects in monolayer cultures to the same extent, polyhedra formation could not be observed. The experiments demonstrate that replication of M. brassicae-NPV as well as 197
Fig. 1. (a) NPV of Autographa californica: cubic inclusion bodies in the nuclei of Mb 0503 cells; 10 mm=19~tm; (b) NPV of Mamestra brassicae: polyhedral inclusion bodies in the nucleus of a Mb 1203 cell; 10mm=13gm
A. caliCbrnica-NPV is possible in the two M. brassicae cell lines tested. In another series of experiments with suspension cultures A. californica-NPV replication was also observed in at least 10 subsequent subcultures of Mb 0503 and Mb 1203. The percentage of infected cells is similar to that measured in the respective monolayer cultures. This work was supported by the Bundesministerium ftir Forschung und Technologie (BMT 15 b). Received December 19, 1975 1. Goodwin, R.H., et al.: J. Invertebr. Pathol. 16, 284 (1970); Ign0ffo, C.M., Shapiro, M., Hink, W.F.: ibid. 18, 131 (1971); Sohi, S.S., Cunningham, J.C.: ibid. 19, 51 (1972); Gardiner, G.R., Stockdale, H. : ibid. 25, 363 (1975) 2. Grace, T.D.C., Brzostowski, H.W.: J. Insect Physiol. 12, 625 (1966) 3. Schneider, I., in: Tissue Culture, Methods and Application, Chapt. 15. New York: Academic Press 1973 4. Ignoffo, C.M.: Bull. Entomol. Soc. Amer. 14, 265 (1968)
2-Phenylethanol Isolated from Bark Beetles J.A.A. Renwick, G.B. Pitman, and J.P. Vit~* Boyce Thompson Institute, Yonkers, New York 10701 The presence of 2-phenylethanol in emergent males of Dendroctonus brevicomis LeConte and feeding males of Ips paraconfusus Lanier has been discovered. Field bioassays indicate that this alcohol is involved in the chemical communication system of L paraconfusus. Gas chromatographic (GC) analyses of hindguts from emergent D. brevicomis revealed the presence of a fragrant-smelling compound in males, which was not detected in females. Hindguts from about 2,000 emergent male beetles were extracted with ethyl ether, and the unknown material was * Present address: Forstzoologisches Institut der Universitfit, Bertoldstr. 17, D-7800 Freiburg i. Br., Federal Republic of Germany. 198
analyzed by combined gas chromatography-mass spectrometry. The mass spectrum indicated a molecular weight of 122, and major fragments at m/e 91 (base peak), 92 and 65 were characteristic of 2-phenylethanol. The mass spectrum and GC retention times (FFAP and OV-1 columns) of an authentic sample of 2-phenylethanol were identical to those of the natural material. A compound with the same GC retention time was detected in the hindguts of L paraconfusus males which had fed in ponderosa pine billets. The identity of this compound as 2phenylethanol was confirmed in the same manner, using hindguts from feeding male beetles as the source of material. No 2-phenylethanol was detected in female beetles. In field bioassays, the response of D. brevicomis to its known attractant (frontalin+exobrevicomin+terpenes) [1, 2] was not significantly affected by the addition of 2-phenylethanol. However, the response of L paraconfusus to male-infested log sections in funnel olfactometers [3] was considerably higher when 2-phenylethanol was added. In bioassays conducted in Grass Valley, California, between 29 June and 10 August, 1975, each of two olfactometers was baited with a ponderosa pine billet in which 25 male L paraconfusus had been feeding for a period of 3 to 5 days. The 2-phenylethanol was dispensed from a 1-mm diameter glass capillary tube held in a horizontal position in one olfactometer for the duration of each daily test (9.00 to 21.00 h). The 2-phenylethanol was switched to the other olfactometer before each new test. The average number of I. paraconfusus responding to the infested billet alone was 78.9, with a range of 32 to 156 for 9 tests, and the sex ratio of responding beetles was 1 :2.0 (males: females). With the addition of 2-phenylethanol, an average of 116.2 beetles, with a range of 45 to 225, were caught, and the sex ratio was 1:2.5. This 47% enhancement of response, along with the production of the compound by the beetles during their stage of highest attraction [3], suggests that 2-phenylethanol plays some role in the aggregation of L paraconfusus. Although three compounds involved in the pheromone system of this beetle have been identified [4], synthetic mixtures have not been as effective as the natural attractant [5]. 2-Phenylethanol is known to be a component of secretions from male scent brushes of certain Lepidoptera [6], but its presence in bark beetles was previously unreported. Supported in part by the Margaret T. Biddle Foundation and National Science Foundation Grant BMS 73-01599. Received January 2, 1976 1. Vit+, J.P., Pitman, G.B.: J. Insect Physiol. 15, 1617 (1969) 2. Bedard, W.D., Silverstein, R.M., Wood, D.L. : Science 167, 1638 (1970) 3. Vit6, J.P., Gara, R.I. : Contrib. Boyce Thompson Inst. 21, 251 (1962) 4. Silverstein, R.M., Rodin, J.O., Wood, D.L.: Science 154, 509 (1966) 5. Wood, D.L., et al. : ibid. 159, 1373 (1968) 6. Aplin, R.T., Birch, M.C. : Experientia 26, 1193 (1970)
Heterochromatin Underreplication in Tropaeolum Embryogenesis W. Nagl, C. Peschke, and R. van Gyseghem Department of Biology, University of Kaiserslautern Heterochromatin underreplication is a phenomenon frequently observed during the polyploidization and polytenizaNaturwissenschaften 63 (1976)
9 by Springer-Verlag 1976
Fig. 1. (a) Lage und Form der Kristallnadeln innerhalb der Oehfmsewand werden durch die organische Hiillsubstanz markiert. Der Calcit wurde w/ihrend der Fixierung herausgel6st. Die Kristalle sind zufallsgem/iB orientiert, eine Matrix zwischen ihnen fehlt. (b) Nur w/ihrend der Geh/iusebildung finden sich im Cytoplasma Vesikel, die jeweils mehrere Kristallnadeln mit ihren Hfillen enthalten. Pr~iparation wie bei (a)
Elektronenmikroskopische Untersuchungen an Calzituba polymorpha Roboz haben nun gezeigt, dab hier die Kristallnadeln der Gehfiusewand in cytoplasmatischen Vesikeln gebildet und erst dann nach augen transportiert und zu einer Wand zusammengeftigt werden (Fig. 1 b). Ffir die Kristallanordnung innerhalb der Wandung ist also nicht eine epitaktische Anlage von Kristallkeimen auf oder in einer Matrix verantwortlich, sondern Transport-Pseudopodien, welche die vorgefertigten KristaUnadeln auf einer organischen Membran weitgehend zufallig anlagern. Calcitkristalle miissen gegeniiber dem umgebenden Medium vor Aufl6sung geschiitzt werden. Bei den bisher untersuchten Foraminiferen ist das Geh/iuse allseits yon einer organischen Schicht fiberzogen. Bei Calzituba I/igt sich eine/iul3ere Htillschicht jedoch nicht nachweisen. Darer ist jede einzelne Kristallnadel in einer organischen Hfille verpackt (Fig. 1). Erstmals zeigen sich hier auch Parallelen in den Morphogeneseablfiufen zwischen einer Foraminifere und den Testaceen, deren Geh/iuse ebenfalls aus im Cytoplasma vorgefertigten Bauelementen (Protein oder Calcit) erstellt wird [3]. Ich danke der Deutschen Forschungsgemeinschaft ffir die finanzielle Unterstfitzung dieser Arbeit (SFB 53). Eingegangen am 27. Januar 1976" 1. Towe, K.M., Cifelli, R.: J. Paleontol. 41, 742 (1967) 2. Haake, F.-W.: J. Foraminiferal Res. 1, 187 (1971) 3. Netzel, H. : Z. Zellforsch. 135, 45 (1972) Naturwissenschaften 63 (1976)
9 by Springer-Verlag 1976
The in vitro replication of nuclear polyhedrosis viruses (NPV) in monolayer cultures of insect cell lines has already been described [1]. This kind of investigation may render new knowledge with regard to the possibility of biological pest control with species-specific viruses since, for example, NPV are responsible for diseases in various lepidopterous larvae like the cotton bollworm (Heliothis zea). Therefore further experiments on in vitro NPV replication are of increasing interest. Five cell lines of the cabbage moth (Mamestra brassicae) were derived from tissue of four/five instar larvae in our laboratory 14-18 months ago: Mb 1203 (mixed explant from the ovary and the dorsal vessel), Mb 0503, Mb 2006, Mb 2007, and Mb 2506 (hemocytes). The cells have so far been subcultured more than a hundred times in a modified Grace-medium [2] and supplemented with 5 % fetal calf serum at 28 ~ in polystyrene plastic flasks (Falcon Plastics; Oxnard, California) or 180-ml glass bottles (Schott, Mainz). The doubling time ranges from 14-36 h depending on the cell line. In the monolayer cultures, most of the predominantly spindlelike cells adhere firmly to the vessel surface. For several months Mb 0503 and Mb 1203 have been growing very well in suspension cultures with a doubling time of 24-26 h. The morphology, culture-, and colony-growth curves as well as chromosome numbers per cell were used as parameters for the regular control of cell-line characteristics. As reported earlier, in somatic lepidopteran tissues, the cells may be highly polyploid or mixoploid with chromosome numbers up to 128 n [3]. Analyses of chromosome preparations from our cell lines showed in several hundred cells mixoploidy. Most cells have many more chromosomes than in the diploid set: 150 250, sometimes more than 300. The chromosomes are, with few exceptions, of minute size but all have obviously diffuse centromeres. We were able to determine the haploid number of meiotic stages of larval testis cells as 1 n = 31. To evaluate if our Mb cell lines would be suitable for NPV replication we infected two of them (Mb 0503, Mb 1203) that proliferated best, with infectious hemolymph of Autographa californica (Alfalfa looper), the NPV of which is known to have a relatively wide host-spectrum specificity [4]. Twelve h after introducing 0.05 ml of a cell- and inclusion body-free supernatant (from hemolymph of heavily infected larvae) into young cultures in both Mb-lines 80 to 90% of the early logphase cells showed cytopathic effects: nuclear granulation and hypertrophy. Two days later in each of these cells up to 100 cubic polyhedra had developed (Fig. 1 a). The centrifuged cell- and polyhedra-free supernatant from these cultures was again infectious to the same extent when introduced into healthy monolayer Mb cultures. We then infected the cell lines with infectious supernatant of centrifuged M. brassicae larval hemolymph. Again after 6-12 h cytopathic effects appeared but to a lessser degree. Ten to 30% of the cells showed alteration of the nuclei and developed up to 30 polyhedral inclusion bodies within the following six days (Fig. 1b). Although the supernatant of these cultures again induces cytopathic effects in monolayer cultures to the same extent, polyhedra formation could not be observed. The experiments demonstrate that replication of M. brassicae-NPV as well as 197
Fig. 1. (a) NPV of Autographa californica: cubic inclusion bodies in the nuclei of Mb 0503 cells; 10 mm=19~tm; (b) NPV of Mamestra brassicae: polyhedral inclusion bodies in the nucleus of a Mb 1203 cell; 10mm=13gm
A. caliCbrnica-NPV is possible in the two M. brassicae cell lines tested. In another series of experiments with suspension cultures A. californica-NPV replication was also observed in at least 10 subsequent subcultures of Mb 0503 and Mb 1203. The percentage of infected cells is similar to that measured in the respective monolayer cultures. This work was supported by the Bundesministerium ftir Forschung und Technologie (BMT 15 b). Received December 19, 1975 1. Goodwin, R.H., et al.: J. Invertebr. Pathol. 16, 284 (1970); Ign0ffo, C.M., Shapiro, M., Hink, W.F.: ibid. 18, 131 (1971); Sohi, S.S., Cunningham, J.C.: ibid. 19, 51 (1972); Gardiner, G.R., Stockdale, H. : ibid. 25, 363 (1975) 2. Grace, T.D.C., Brzostowski, H.W.: J. Insect Physiol. 12, 625 (1966) 3. Schneider, I., in: Tissue Culture, Methods and Application, Chapt. 15. New York: Academic Press 1973 4. Ignoffo, C.M.: Bull. Entomol. Soc. Amer. 14, 265 (1968)
2-Phenylethanol Isolated from Bark Beetles J.A.A. Renwick, G.B. Pitman, and J.P. Vit~* Boyce Thompson Institute, Yonkers, New York 10701 The presence of 2-phenylethanol in emergent males of Dendroctonus brevicomis LeConte and feeding males of Ips paraconfusus Lanier has been discovered. Field bioassays indicate that this alcohol is involved in the chemical communication system of L paraconfusus. Gas chromatographic (GC) analyses of hindguts from emergent D. brevicomis revealed the presence of a fragrant-smelling compound in males, which was not detected in females. Hindguts from about 2,000 emergent male beetles were extracted with ethyl ether, and the unknown material was * Present address: Forstzoologisches Institut der Universitfit, Bertoldstr. 17, D-7800 Freiburg i. Br., Federal Republic of Germany. 198
analyzed by combined gas chromatography-mass spectrometry. The mass spectrum indicated a molecular weight of 122, and major fragments at m/e 91 (base peak), 92 and 65 were characteristic of 2-phenylethanol. The mass spectrum and GC retention times (FFAP and OV-1 columns) of an authentic sample of 2-phenylethanol were identical to those of the natural material. A compound with the same GC retention time was detected in the hindguts of L paraconfusus males which had fed in ponderosa pine billets. The identity of this compound as 2phenylethanol was confirmed in the same manner, using hindguts from feeding male beetles as the source of material. No 2-phenylethanol was detected in female beetles. In field bioassays, the response of D. brevicomis to its known attractant (frontalin+exobrevicomin+terpenes) [1, 2] was not significantly affected by the addition of 2-phenylethanol. However, the response of L paraconfusus to male-infested log sections in funnel olfactometers [3] was considerably higher when 2-phenylethanol was added. In bioassays conducted in Grass Valley, California, between 29 June and 10 August, 1975, each of two olfactometers was baited with a ponderosa pine billet in which 25 male L paraconfusus had been feeding for a period of 3 to 5 days. The 2-phenylethanol was dispensed from a 1-mm diameter glass capillary tube held in a horizontal position in one olfactometer for the duration of each daily test (9.00 to 21.00 h). The 2-phenylethanol was switched to the other olfactometer before each new test. The average number of I. paraconfusus responding to the infested billet alone was 78.9, with a range of 32 to 156 for 9 tests, and the sex ratio of responding beetles was 1 :2.0 (males: females). With the addition of 2-phenylethanol, an average of 116.2 beetles, with a range of 45 to 225, were caught, and the sex ratio was 1:2.5. This 47% enhancement of response, along with the production of the compound by the beetles during their stage of highest attraction [3], suggests that 2-phenylethanol plays some role in the aggregation of L paraconfusus. Although three compounds involved in the pheromone system of this beetle have been identified [4], synthetic mixtures have not been as effective as the natural attractant [5]. 2-Phenylethanol is known to be a component of secretions from male scent brushes of certain Lepidoptera [6], but its presence in bark beetles was previously unreported. Supported in part by the Margaret T. Biddle Foundation and National Science Foundation Grant BMS 73-01599. Received January 2, 1976 1. Vit+, J.P., Pitman, G.B.: J. Insect Physiol. 15, 1617 (1969) 2. Bedard, W.D., Silverstein, R.M., Wood, D.L. : Science 167, 1638 (1970) 3. Vit6, J.P., Gara, R.I. : Contrib. Boyce Thompson Inst. 21, 251 (1962) 4. Silverstein, R.M., Rodin, J.O., Wood, D.L.: Science 154, 509 (1966) 5. Wood, D.L., et al. : ibid. 159, 1373 (1968) 6. Aplin, R.T., Birch, M.C. : Experientia 26, 1193 (1970)
Heterochromatin Underreplication in Tropaeolum Embryogenesis W. Nagl, C. Peschke, and R. van Gyseghem Department of Biology, University of Kaiserslautern Heterochromatin underreplication is a phenomenon frequently observed during the polyploidization and polytenizaNaturwissenschaften 63 (1976)
9 by Springer-Verlag 1976
