Effect of cycloheximide on different stages of Drosophila melanogaster

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<ul><li><p>Toxicology Letters, 13 (1982) 105-112 Elsevier Biomedical Press </p><p>105 </p><p>EFFECT OF CYCLOHEXIMIDE ON DIFFERENT STAGES OF </p><p>DROSOPHILA MELANOGASTER </p><p>R. MARCOS, J. LLOBERAS, A. CREUS, N. XAMENA and 0. CABRB </p><p>Departamento de Gent!tica, Fact&amp;ad de Ciencias, Universidad Autdnoma de Barcelona, Bellaterra </p><p>(Spain) </p><p>(Received September lOth, 1981) (Revision received April 3rd, 1982) (Accepted April Sth, 1982) </p><p>SUMMARY </p><p>Cycloheximide, an antibiotic inhibiting protein synthesis, exerted a toxic effect on different developmental stages egg, larva and adult of Drosophila melanogaster. At the egg stage the early embryos were most sensitive. With larvae, a strong decrease in viability was found, with no sex difference. In adults, there was a dose-effect relationship, mortality increasing with concentration. At 10 and 15 mM, males were more sensitive than females. </p><p>There were consistent differences between the control and cycloheximide-fed females in respect of the average number of eggs deposited and offspring produced. </p><p>INTRODUCTION </p><p>The antibiotic cycloheximide (or actidione) [l] inhibits protein synthesis at the translation level [2, 31, acting exclusively on cytoplasmic (80s) ribosomes of eukaryotes [4]. </p><p>All the energy-dependent stages in the protein-synthesizing process are affected, although initiation seems the most sensitive [5, 61. Cycloheximide also affects respiration [7], ion uptake [S], amino acid biosynthesis [9], and DNA and RNA synthesis [lo], effects that are probably secondary to its effect on protein synthesis. </p><p>In Drosophila cycloheximide affects the functional morphology of polytene chromosomes of salivary glands [l l] and their synthesizing activity [12]. </p><p>It has been suggested that cycloheximide can suppress a long-lasting modification of a phototactic behaviour in Drosophila [ 131. We report a series of experiments in which we have tested the effect of cycloheximide on different developmental stages in D. melanogaster: viz. egg, larva and adult. </p><p>0378-4274/82/0000-0000/$02.75 0 Elsevier Biomedical Press </p></li><li><p>106 </p><p>MATERIALS AND METHODS </p><p>A Berlin-K stock (wild-type strain) of D. melanogaster was used. The flies were provided from mass cultures that had been reared in plexiglass cage populations maintained in our laboratory at 25 +_ 1 C. All procedures were performed at this temperature. </p><p>The cycloheximide was purchased from Sigma (St. Louis, MO). </p><p>Eggs. 3% agar plates (5 x 20 cm) coated with a paste of yeast and grape juice were placed in the population cages for 1 h. After the laying period, the plates were rinsed with water and the eggs washed off and trapped in a plastic cylinder one end of which was covered with a 250 mesh screen. The eggs were washed thoroughly with 0.12 M NaCl, 0.1% Triton X-100 solution and finally with 0.12 M NaCl [14, 151, dechorionized by immersion in 2% sodium hypochlorite for 2 min and washed again with saline [ 161. </p><p>The vitelline membrane was rendered permeable by means of heptane vapours; the cylinder with the trapped eggs was placed 1 cm over the heptane surface into a closed beaker for 30 min and the vapour removed by draft. </p><p>Eggs were incubated in Zalokars medium [17] with different cycloheximide concentrations for 30 min, washed with the medium and incubated in microtest plates. Scores were recorded of the cleavage number (syncytium), blastoderm and post-blastoderm stages, which are easily visible at x 100 magnification [15]. </p><p>At 30-min intervals up to 4 h after cycloheximide treatment, the eggs were restored. 24 h later the number of non-eclosioned embryos was counted. For each cycloheximide concentration 400 eggs were scored. The control eggs were washed and manipulated in an identical way. </p><p>Larvae. Petri dishes containing agar were introduced into the population cages for 3 h. At the end of the laying period, the eggs deposited were counted and put into culture vials (100 eggs/vial) containing 25 ml of standard food medium enriched with living yeast to stimulate feeding of the larvae. After 24 h (when the vials contained first-instar larvae) 1 ml of test solution of cycloheximide containing 5% w/v sucrose was added to each vial. Controls were treated with 5% sucrose only. Surviving adults were counted and sexed. </p><p>Adults. 3-day-old males and females (200/sex/experiment), were put in special feeding units [18] and given different concentrations of cycloheximide in 5% sucrose. Dead flies were counted every 12 h for 4 days. Control flies were fed with 5% sucrose only. </p><p>Egg and offspring production. After treatment, virgin females were crossed individually with untreated males of the same age in small vials containing 8 ml of standard medium for 3 days, using 50 replicates/group. On the fourth day each surviving pair was transferred into new vials open at both ends. A cotton plug was placed in the upper vial and nutrient agar (diluted with grape juice) seeded with live yeast in the lower one. The agar was removed and replaced daily and the eggs laid </p></li><li><p>107 </p><p>were counted and kept at 25C and the number of offspring produced were recorded. </p><p>RESULTS AND DISCUSSION </p><p>With the egg, there was 11% retention (the average number of eggs in post- blastodermal stages at the moment of scoring). </p><p>Egg viability was unaffected by heptane vapour, although a slight delay in development was observed. </p><p>Cycloheximide affected development during the first hours (Fig. l), there being a gradual decrease in the number of embryos that passed the blastoderm stage with increasing concentration of cycloheximide. Furthermore, the non-eclosioned eggs at 24 h reached a maximum (approx. 90070) starting from 1 mM cycloheximide (Fig. 2) </p><p>OmM </p><p>0.2 mM </p><p>0.5 mM </p><p>In-It4 </p><p>3mM </p><p>Fig. 1. Percentage of embryos that overcome the blastoderm stage after cycloheximide treatment. </p></li><li><p>108 </p><p>0.2 0.5 1 3 </p><p>Concentratbon (ml4 ) </p><p>Fig. 2. Percentage of non eclosioned eggs 24 h after cycloheximide treatment. </p><p>up to 17 mM, the highest concentration tested, but in this range the deviations did not attain statistical significance. </p><p>These results indicated that in our experimental conditions there was a diffusion effect of cycloheximide inside the egg, a minimum concentration equivalent to 1 mM being necessary for maximal effect. Surviving embryos were unaffected if they had reached a certain degree of differentiation, but the early embryos, with a fast cellular cycle (10 min at 25 C [IS], were sensitive. </p><p>Cycloheximide produced a strong decrease in the viability of larvae (Table I). The data were corrected for control lethality by the use of Abbotts formula [19]. </p><p>At the concentrations tested, there were no significant differences in sex ratio between cycloheximide-treated and control larvae. Nigsch et al. (201 reported equal sensitivity of both sexes for the same strain (Berlin wild) subjected to caffeine. </p><p>There was a low viability in the control population: Chapco [21] postulates that it is net fitness which determines the survival of a genome. A high fecundity and a </p><p>TABLE I </p><p>PREIMAGINAL LETHALITY AFTER TREATMENT OF LARVAL POPULATIONS OF </p><p>DROSOPHILA MELANOGASTER WITH CYCLOHEXIMIDE </p><p>Concentration Eggs </p><p>(mM) counted </p><p>Number of flies emerged Ratio Lethality (070) </p><p>Males Females Total 9:cY </p><p>Observed Induceda </p><p>0 7ooo 1564 1623 3187 1.04 54.48 - </p><p>1 7100 542 525 1067 0.97 84.97 66.98 </p><p>3 7100 317 306 623 0.96 91.23 80.71 </p><p>aFor the calculation of induced lethality Abbotts correction was used. </p></li><li><p>109 </p><p>low viability may be sufficient to ensure survival in a particular set of environmental conditions, e.g.: high competition in population cages. </p><p>Concentration-mortality relationships in adults express the biological reactivity of chemicals tested. Fig. 3 shows induced mortalities calculated from the equation: </p><p>M (90) = 100 - St/.sc x 100, corrected for death resulting from causes other than cycloheximide treatment and plotted against exposure time [22], where St = percentage survival for the treated groups and sc = percentage survival for the control. These results indicate that there was a dose-effect relationship, in which mortality increased with the concentration. The sensitivity differences between sexes, less evident at low concentrations, became clear at high concentrations (10 and 15 mM). </p><p>An increased sensitivity of males with respect to caffeine [23,24] or MMS [25,26] had been shown with other stocks. </p><p>- Males IOO- </p><p>- - Females - - - </p><p>go- </p><p>80- </p><p>70- </p><p>40- </p><p>30- </p><p>12 24 36 46 60 72 84 96 </p><p>Fig. 3. Exposure-mortality relationships to cycloheximide of D. melunogaster males and females during adult feeding. </p></li><li><p>110 </p><p>TABLE II </p><p>EGG AND OFFSPRING PRODUCTION OF CONTROL AND TREATED FEMALES OF </p><p>DROSOPHILA MELANOGASTER BETWEEN THE 4th AND 7th DAYS AFTER CYCLO- </p><p>HEXIMIDE TREATMENT </p><p>4 5 6 I </p><p>Control (a) Vials 37 34 31 2s </p><p>Eggs per vial 44.59 41.29 37.67 35.84 </p><p>Progeny per vial 27.38 22.47 21.70 19.68 </p><p>Viability (070) 61.39 54.41 57.61 54.91 </p><p>1 mM (b) Vials 8 8 7 I </p><p>Eggs per vial 6.62 14.25 28.42 32.57 </p><p>Progeny per vial 3.50 7.00 17.00 19.57 </p><p>Viability (%) 52.83 49.12 59.79 60.08 </p><p>Ratio (b): (a) 0.12 0.31 0.78 0.99 </p><p>3 mM (c) Vials 11 11 11 11 </p><p>Eggs per vial 2.63 5.72 14.00 16.27 </p><p>Progeny per vial 1.45 2.09 9.18 12.90 </p><p>Viability (%) 55.17 36.50 65.58 79.32 </p><p>Ratio (c): (a) 0.05 0.09 0.64 0.66 </p><p>Egg laying may be equated with general metabolic level. Table II shows the egg and offspring production of control and treated (1 and 3 mM) females. All surviving females at 10 and 15 mM died before determination of egg and offspring </p><p>production. There was evidence of decreased fecundity and fertility in the treated flies. The </p><p>ratio between the mean number of progeny per vial in the treated and in the control series provides a good estimate of the fertility of the females treated as adults with cycloheximide. This ratio increased from a low value at the fourth day after treatment approaching the control value in the 1 mM series by the end of the test, and reaching an intermediate value in the 3 mM series. The gradual recuperation of fertility suggested a detoxication process. </p><p>Treatment of females with cycloheximide did not affect the viability of the eggs laid, from when it would seem that cycloheximide has a quantitative metabolic effect, decreasing the pool of essential substances for egg production, but not affecting qualitatively substances required for egg development. </p><p>It may be concluded that cycloheximide is toxic to D. melanogaster at all stages, but more so at those characterized by high metabolic activity, viz. pre-imaginal stages and egg production. This agrees with the role of cycloheximide as an inhibitor of protein synthesis. </p></li><li><p>111 </p><p>REFERENCES </p><p>1 P. Sentein, Nuclear and mitotic abnormalities produced by cycloheximide in the newt egg during cleavage and their relationship to the cell cycle, Exp. Cell Biol., 49 (1981) 98-l 17. </p><p>2 P. Sentein, Action de la cycloheximide sur les noyaux de loeuf de triton au debut de la segmentation, Arch. Biol. Bruxelles, 87 (1976) 43-68. </p><p>3 M. Ronne, In vitro induction of uncoiled chromosomes, Hereditas, 86 (1977) 11 l-l 14. 4 S. Pestka, Inhibitors of ribosome functions, Annu. Rev. Biochem., 40 (1971) 487-562. 5 T.G. Cooper and J. Bossinger, Selective inhibition of protein synthesis initiation in Sacchoromyces </p><p>cerevisiue by low concentrations of cycloheximide, J. Biol. Chem., 251 (1976) 7278-7280. 6 N.L. Oleinick, Initiation and elongation of protein synthesis in growing cells: differential inhibition </p><p>by cycloheximide and emetine, Arch. Biochem. Biophys., 182 (1977) 171-180. 7 S.B. Wilson and A.L. Moore, The effects of protein synthesis inhibitors on oxidative </p><p>phosphorylation by plant mitochondria, Biochim. Biophys. Acta, 292 (1973) 603-610. 8 I.R. Mac Donald and R.J. Ellis, Does cycloheximide inhibit protein synthesis specifically in plant </p><p>tissues?, Nature, 222 (1969) 791-792. 9 A. Oaks and F.J. Johnson, The effect of cycloheximide on amide formation in maize roots, Can. J. </p><p>Bot., 51 (1973) 91-95. 10 J.L. Farber and R. Farmar, Differential effects of cycloheximide on protein and RNA synthesis as a </p><p>function of dose, Biochem. Biophys. Res. Commun., 51 (1973) 626-630. 11 N. Mitra and A.S. Murkherjee, Analysis of functional morphology of polytene chromosomes of </p><p>Drosophila melanoguster after treatment with cycloheximide, Proc. Indian Sci. Congr., 60 (1973) 431. </p><p>12 H. Schoon and L. Rensing, Effects of protein synthesis inhibiting antibiotics of the puffing pattern of Drosophila salivary gland chromosomes in vitro, Cell. Diff., 2 (1973) 97-106. </p><p>13 R. Willmun, H. Broda and K.P. Sieber, Cycloheximide suppresses a behavioural modification in Drosophila, Naturwissenschaften, 66 (1979) 318-319. </p><p>14 H.J. Kriegstein and D.S. Hognes, Mechanism of DNA replication in Drosophila chromosomes: structure of replication forks and evidence for bidirectionality, Proc. Natl. Acad. Sci. USA, 71 (1974) 135-139. </p><p>15 M. Bownes, A photographic study of development in the living embryos of Drosophila melunoguster, J. Embryol. Exp. Morphol., 33 (1975) 789-801. </p><p>16 D.L. Hill, Chemical removal of the chorion from Drosophila eggs, Drosophila Inf. Serv., 19 (1945) 62. </p><p>17 M. Zalokar, Autoradiographic study of protein and RNA formation during early development of Drosophila eggs, Develop. Biol., 49 (1976) 425-437. </p><p>18 E. Vogel and H. Ltiers, A comparison of adult feeding to injection in Drosophila melunogaster, Drosophila Inf. Serv., 51 (1975) 113-114. </p><p>19 W.S. Abbott, A method for computing the effectiveness of an insecticide, J. Econ. Entomol., 18 (1925) 265-267. </p><p>20 J. Nigsch, l-l. Graf and F.E. Wurgler, Caffein toxicity in Drosophila strains having different MMS sensitivities, Mutation Res., 43 (1977) 57-64. </p><p>21 W. Chapco, Correlations between chromosome segments and fitness in Drosophila melunoguster, II. The X chromosome and egg viability, Genetics, 92 (1979) 595-601. </p><p>22 E. Vogel and A.T. Natarajan, The relation between reaction kinetics and mutagenic action of mono- functional alkylating agents in higher eukaryotic systems, I. Recessive lethal mutations and translocations in Drosophila, Mutation Res., 62 (1979) 51-100. </p><p>23 W. Kuhlmann, H.G. Fromme, E.M. Heege and W. Ostertag, The mutagenic action of caffeine in higher organisms, Cancer Res., 28 (1968) 2375-2389. </p></li><li><p>112 </p><p>24 S. Zimmering, R. Kofkoff and C. Osgood, Survival of caffeine-fed adult males and females from </p><p>strains of Drosophila melanogaster, Mutation Res., 43 (1977) 453-456. </p><p>25 M. Gatti, S. Pimpinelli, A. de Marco and C. Tanzarella, Chemical induction of chromosome </p><p>aberrations in somatic cells of Drosophila melanogaster, Mutation Res., 33 (1975) 201-212. </p><p>26 U. Graf and F.E. Wtirgler, MMS-sensitive strains in Drosophila melanogaster, Mutation Res., 34 </p><p>(1976) 251-258. </p></li></ul>

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