Male diploid embryonic cell line ofDrosophila virilis

Summay A new established cell line 79f7Dv3g, ofDrosophila virilis consisting initially of male and female cells and represented now, after 6 yr of cultivation, only by male cells is described. The population doubling time is 36 h at 25° C. The cell culture is also able to grow in serum-free media for an indefinite time without special selection and has a population doubling time of 2 d.     

Bristle patterns,clones and cell competition along the anterior margin ofNotch wings ofDrosophila hydei

Summary The bristle pattern along the anterior margin ofNotch (N1-22.3) wings ofDrosophila hydei and the occurrence ofyellow (y 1–38.8) marked clones induced by X-ray irradiation during various larval stages are described. UnirradiatedN/N ⁺ wings show gaps (notches) in the longitudinal bristle rows along the 1st longitudinal vein, with tufts of bristles particularly near gaps. X-ray irradiation increases the number and total length of the gaps. The patterning of bristles along the margin depends on theN ⁽⁺⁾ genotype of the induced clones. RecombinantN ⁺/N ⁺ clones from irradiated wings show excessive growth with an autonomous wildtype bristle pattern. Characteristically, these clones do not respect the dorso-ventral compartment boundary along the wing margin, do not follow an exponential (2ⁿ) growth pattern, tend to fill the gaps with bristles and theiryellow medial row bristles are less often interspersed withy ⁺ bristles than described forN ⁺/N ⁺ wings. HomozygousN appears to be a cell lethal condition inD. hydei as it is inD. melanogaster. When y clones were kept phenotypicallyNotch (viz.,N/N/N ⁺) as the background cells, we found a lower number ofy bristles, a lower percentage of mosaic wings but also a reltive deficiency ofy ⁺ interspersions. The latter is discussed in relation to a possible clonal originof the notches.     

Bristle patterns and clones along a compartment border in the anterior wing margin ofDrosophila hydei

Summary The bristle pattern along the first longitudinal vein of the wing ofD. hydei differs from that ofD. melanogaster. Instead of a triple row,D. hydei and some allied species show a pattern of five parallel bristle rows of which the medial row (MR) is comparable to the medial triple row (MTR) ofD. melanogaster. Cells of the MR can be made homozygousyellow (y) by induction of mitotic recombination in heterozygousy/y ⁺ females. Until 70 h after egg laying (AEL), the MR clones inD. hydei overlap with one or more of the accompanying dorsal and ventral bristle rows. Between 70 and 120 h AEL the MR clones only overlap with dorsal bristle rows. Some time later they also become separated from both dorsal rows. The resulting MR clone pattern fits with the overall longitudinal clone pattern in the wing blade ofD. melanogaster described by Bryant (1970) and others. The MR clones inD. hydei, however, often show a fragmented appearance with many indentations of the surroundingy ⁺ tissue even when induced after fixation of the DV compartment boundary. This result contrasts with the commonly held notion, derived from work withD. melanogaster, that compartment boundaries are smooth lines.     

Cell lines from imaginal discs ofDrosophila melanogaster

Summary New cell lines, designated as ML-DmDl≈10, were established from dissociated imaginal discs ofDrosophila melanogaster. The culture medium was prepared by mixing in a 1:1 ratio Cross and Sang’s M3(BF) medium, supplemented with 10% heat inactivated fetal bovine serum (FBS), with the supernatant of a primary embryonic cell culture made in the M3(BF) medium and supplementing this mixture with insulin. One cell line was established in the medium containing larval hemolymph instead of the primary culture supernatan, and another was established in fresh M3(BF) medium supplemented with insulin and FBS. In these mediums, imaginal disc cells first formed aggregates and cellular vesicles within a few weeks followed by the proliferation of thin-layered cells around them after about 1 mo. Ten cell lines have so far been established from two kinds of imaginal discs and disc mixtures. The ploidy of these cell lines was predominantly diploid. Population doubling time was about 50 to 70 h at 3 to 10 mo. after initiation of the culture. When the cell aggregates formed in vitro were implanted in metamorphosing larvae, they differentiated at high frequency into adult cuticular strutures in the early phase of the primary culture. This differentiation of aggregates was also observed, though at low frequency, in a culture maintained by dilution-transfer for 6 to 15 mo. in vitro.     

Contamination and persistent infection ofDrosophila cell lines by reovirus type particles

Summary A new type of contaminant particles persistently infectedDrosophila cell lines. On an ultrastructural, morphogenetic and histochemical basis, they are similar to viruses of the Reoviridae group. They have been namedDrosophila K virus (DKV).     

Partial hyperploidy of the X-chromosome inDrosophila hydei leading to duplication of male gonadal and genital structures

Summary Introduction of two doses of the X-chromosomalw ^mCo duplication next to a normal X-chromosome in males ofD. hydei leads to duplication of testis tissue and structures derived from the male genital disc. The effect of this partial hyperploidy of the X-chromosome seems restricted to the male. We tentatively conclude that this part of the X-chromosome contains some factor(s) which may specifically affect the reproductive system and analia of males.     

The development of a medium supplemented with egg extract for the maintenance ofDrosophila cell lines

Summary A saline extract was prepared fromDrosophila eggs. When diluted to a concentration of 1% withDrosophila tissue culture medium, it did not support growth of cells from theDrosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time fourDrosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonicDrosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells. This work was supported by a grant from the Science Research Council of Great Britain.     

Characterization of invertebrate cell lines

Summary Cell extracts of five mosquito cell lines and a tick cell line were examined for four cellular isozymes using a cellulose-acetate electrophoretic technique. This method distinguished the cell lines that were derived from the different species. Intraspecies distinctions were not made using the cell lines tested; the significance of this finding is discussed. The usefulness of this technique in identifying a potentially mislabeled cell line was demonstrated. This research was supported by contracts, DADA 17-72C-2170 of the U.S. Army and N00014-78C-0104 of the U.S. Office of Naval Research and grants from the World Health Organization and the Rockefeller Foundation.     

Rudimentary mutants ofDrosophila melanogaster

Summary The X-linkedrudimentary (r) mutants ofDrosophila melanogaster are pyrimidine auxotrophs and require exogenous pyrimidines (Nørby, 1970; Falk, 1976). We have established a set ofrudimentary cell lines that are derived from embryos, homozygous for eitherr ¹ orr ³⁶. The enzymatic activities of the pyrimidine synthesizing enzymes were measured in the mutant lines. We have further investigated the nutritional requirements of the mutant cells in vitro by using a pyrimidine free culture medium.Ther ¹ cell lines were found to express 3–7%dihydroorotase (DHOase) activity as compared to a wildtype cell line. Reducedaspartate transcarbamylase (ATCase) activity was measured in somer ¹ cell lines whereas wildtypecarbamylphosphate synthetase (CPSase) activity is expressed in allr ¹ cell lines. Ther ³⁶ cell line expresses wildtype activity ofDHOase andCPSase. ATCase activity was found to be reduced to 10% of the wildtype activity.The mutant cell lines do not proliferate in pyrimidine free minimal medium and cell proliferation is obtained by the addition of crude RNA. Proliferation of ther ¹ cells is restored by the supplementation of the minimal medium withdihydroorotate whereas proliferation of ther ³⁶ cells is restored by supplementation with eitherdihydroorotate orcarbamylaspartate.The results demonstrate that therudimentary phenotypesr ¹ andr ³⁶ are expressed at the cellular level and that the two mutant cell types behave as cellular pyrimidine auxotrophs in vitro.     

A study of the female germ line in mosaics ofDrosophila

Summary Our report presents an analysis of the development and dynamics of the female germ line inDrosophila. Females were produced that were mosaic either for attached-X chromosomes and a ring-X (triplo-X-diplo-X), or for and a marked Y-chromosome . The germ-line and genitalia of these females were analysed by direct microscopic observation or by examination of the progeny.Eggs derived from triplo-X germ cells were hardly capable of supporting development, with most of the zygotes dying during embryonic development. The analysis of the germ line was therefore carried out mainly by direct observation of histochemically stained developing oocytes in the ovaries of mosaic females.The total germ cell population of both ovaries of a female was mosaic in 22–29% of the tested animals. From this frequency of mosaicism we estimated the number of functional primordial germ cells to be betwen 3 and 6 cells at the blastoderm stage. At this stage the cell lineages for the left and right ovary are not yet separated.The germ cell population of individual ovarioles was frequently mosaic which shows that the few stem cells in an ovariole are recruited as a group and are not clonal descendants of a single ancestor cell per ovariole. An analysis of the sequential pattern of oocyte-nurse cell cysts in mosaic ovarioles revealed that neighbouring cysts tend to be of the same genotype. This suggests that the stem cells of the adult ovaries preferentially divide in bursts, one of them giving rise to two, three and sometimes even more cystocytes in a row.In addition, the foci for lethality and sterility of the triplo-X condition were determined. Non-mosaic triplo-X females (metafemales) are hardly viable and invariably sterile. Using our mosaics, the focus forlethality could be mapped to a region very near the ventral prothoracic discs. The focus forsterility resides in the genitalia, since flies with triplo-X genitalia never laid any eggs, regardless of the genotype of their ovaries.     

Established cell lines from the beetleDiabrotica undecimpunctata (Coleoptera: Chrysomelidae)

Summary Two new cell lines, designated IPLB-DU182A and IPLB-DU182E, were developed from embryos of the southern corn rootworm,Diabrotica undecimpunctata. Cells were grown in the lepidopteran cell culture media IPL-52B and IPL-76 in a 3∶1 ratiowith 9% fetal bovine serum. The IPL-52B was modified by deleting CaCl₂·2H₂O and NaHCO₃ out of the formulation. The osmotic pressure was adjusted to the optimal osmolarity of 400 mOsm/kg by the addition of2 g mannitol/100 ml medium. The cells were primarily epithelial-like, but some spindle-shaped cells were also present. The lines were 65% diploid and were characterized with respect to 10 isozymes. Cellscurrently grow with a 5-d doubling time and are subcultured by trypsinization at 1-wk intervals and at a 1∶2 to 1∶5 split ratio.     

Spontaneous recombination in males ofDrosophila bipectinata

Spontaneous recombination in males ofDrosophila bipectinata was tested in five wild type laboratory stocks of different geographic origins by using sepia eye and black body colour double recessive mutant stock. The results indicate thatDrosophila bipectinata exhibits spontaneous male recombination. Further, recombination occurs at low rate and there is interstrain variation with respect to the rate of male crossing-over. This is the first report of spontaneous recombination in males ofDrosophila bipectinata.     

Characterization and culture of virus replicating continuous insect cell lines from the bollworm,Heliothis zea (boddie)

Summary A series of five discrete virus replicating insect cell lines were isolated from the ovarian and fat body tissues ofHeliothis zea pupae. Two of these cell lines (IPLB-HZ-1075 and-HZ-1079) were studied in depth as to their growth and virus replication responses to specific nutrients (acetyl-β-methylcholine, fresh glutamine) in a number of media. The same two cell lines were identified to species by serological (microimmunodiffusion) and isozyme (phosphoglucoisomerase and peptidase:glycyl-leucine) techniques. Distinguishing comparisons were made with other cell lines that have been confused with the present lines in the literature and with cell line and host pupal extracts from the same and other lepidopteran species studied concurrently in this laboratory. Sterility culture tests were negative for mycoplasmas. The present fiveH. zea lines were the first insect cell lines to replicate polyhedra from a unicapsid multiple embedded nuclear polyhedrosis virus (Baculovirus Group A), in this case the homologous virus obtained from larvae ofH. zea.     

Endogenous viruses ofDrosophila melanogaster cell lines: Their frequency,identification and origin

Summary Viruses were detected in 32 of the culture medium supernates from 42Drosophila melanogaster cell lines. The picornaviruses DCV, DPV and DAV, which also have been found in natural and laboratory populations of flies, were detected as well as the diplornavirus DXV which has never been noted inDrosophila stocks. Two of the 21 samples of commercial fetal bovine sera that were examined were found to be capable of causing infections of DXV in virus-freeDrosophila flies. It is inferred that the insects used to initiate the cell lines, the sera used in the culture media, and accidental contaminations account for the presence of viruses in the continously culturedDrosophila cell lines.     

Detection of clones in the nervous system ofDrosophila melanogaster

Summary Mitotic recombination was induced, by X-irradiation at the blastoderm stage, in flies heterozygous for one of the temperature-sensitive paralytic mutationsshibire andtp-2. The results show that these mutations can be used to detect the presence of clones in the central nervous system through the temperature-sensitive paralysis of individual legs. Mitotic recombination can also be used to examine the effects of these mutations in the peripheral nervous system; shibire is thus shown to affect the function of sensory neurons.     

The acentriolar state of the Drosophila cell lines 1182

A Drosophila melanogaster cell line devoid of centrioles has been recently described. In order to achieve an easier characterization of these acentriolar cells, we used the monoclonal antibody Bx 63 of M. Frasch which recognizes the Drosophila centrosome. Although centrosomes are detected at every mitotic pole in Drosophila cells with centrioles, no such structure has been observed in 1182-4 acentriolar cells. The antigenic material is, however, present in these cells. Moreover, we noticed a certain proportion of acentriolar cells in 4 other 1182 lines. The lack of centrioles previously found only in the 1182-4 cells seems therefore not accidental and should be linked to their particular origin.