MUTATIONAL RESPONSE OF HABROBRACON OOCYTES IN METAPHASE AND PROPHASE TO ETHYL METHANESULFONATE AND NITROGEN MUSTARD

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1 MUTATIONAL RESPONSE OF HABROBRACON OOCYTES IN METAPHASE AND PROPHASE TO ETHYL METHANESULFONATE AND NITROGEN MUSTARD E. A. L6BBECKE1 AND R. C. VON BORSTEL Biology Division, Oak Ridge National Laboratory,Z Oak Ridge, Tennessee Received March 14, 1962 HITING (1945), working on the parasitic wasp Habrobracon, developed a timing method that distinguished oocytes irradiated in the first meiotic metaphase (metaphase I) from those irradiated in the first meiotic prophase (prophase I). She used embryo lethality of X-irradiated eggs from diploid virgins, which measured mostly dominant lethality, and found that metaphase I oocytes are approximately 20 times as sensitive to X-radiation as prophase I oocytes. When methods for determining recessive lethal frequency for the entire genome of Habrobracon were established (HEIDENTHAL 1952), it was found that metaphase I is also 20 times as sensitive when recessive lethality is the criterion (ATWOOD, VON BORSTEL and WHITING 1956; WHITING and MURPHY 1956). The egg of Habrobracon has such a thin chorion that the stage of embryonic death can be recorded when hatchability is scored (VON BORSTEL and REKEMEYER 1959). With stage of death data available, dominant lethality can be partitioned into different types. The actions of mutagenic agents can be compared with respect to frequencies of early death, which is believed to be associated with mitotic failure (VON BORSTEL 1960, 1961a,b), and later deaths, which derive from a loss of chromosomes or their parts (VON BORSTEL and REKEMEYER 1959). Nitrogen mustard [methyl bis ( p-chloroethyl) amine hydrochloride] has been used on the different stages of oogenesis in Habrobracon, and a 20-fold difference in sensitivity between metaphase I and prophase I for embryo lethality, like that of X-radiation, obtains with this agent ( VON BORSTEL 1955a). Neither recessive lethality nor stages of death were scored accurately at that time, but it was noted that the frequency of early deaths induced by nitrogen mustard is higher, relative to the mean lethal dose, than that induced by X-radiation (cf. VON BORSTEL ). Nitrogen mustard is an alkylating agent which reacts with purine and pyrimidine bases (LAWLEY 1957; LAWLEY and WALLICK 1957; BROOKES and LAWLEY 1960); since the molecule has two reactive arms, cross-linking of polynucleotide chains of deoxyribonucleic acid (DNA) has also been suggested as a source of its mutagenic and/or lethal action (GOLDACRE, LOVELESS and ROSS 1949; LOVELESS 1959; BROOKES and LAWLEY 1961 ). The mutagenic action of ethyl methanesulfonate (EMS) has been observed in a number of organisms, including bacteriophage (LOVELESS, 1958, 1959), E. coli 1 Postdoctoral Fellow of the Deutsche Forschungsgemeinschaft. 2 Operated by Union Carbide Corporation for the U. S. Atomic Energy Commission. Genetics 47: July 1962.

2 854 E. A. LOBBECKE AND R. c. VON BORSTEL ( STRAUSS 1961 ), Neurospora ( KOLMARK, cited by WESTERGAARD 1957), and in Drosophila (FAHMY 1961; FAHMY and FAHMY 1961). EMS can act directly with genetic material, as shown by its mutagenic effect on extracellular phage in experiments in which an indirect action involving other cellular constituents was ruled out (GREEN and KRIEG 1961). Genetic fine structure studies show that it induces point mutations at many sites within a genetic locus (BENZER 1961). EMS reacts with purines and pyrimidines to produce ethylated base analogues ( LAWLEY and WALLICK 1957; PAL, unpublished results), and presumably one or more of these products, produced within exposed DNA ( BROOKES and LAWLEY 1961) is responsible for the induced mutations. Preliminary results in a comparison of EMS-induced revertibility of various point mutations of phage show a high degree of genetic specificity, possibly indicating that EMS-induced mutations are predominantly due to one or a few kinds of base pair substitutions (GREEN and KRIEG 1961; KRIEG, unpublished results; see also FREESE 1961). In tests reported here, the incidence of total embryo lethality (principally dominant lethality), the stages of death, and recessive embryo lethality were measured after metaphase I and prophase I oocytes were exposed to EMS and nitrogen mustard. MATERIALS AND METHODS For all experiments, virgins of the wild stock No. 33 were used. The animals were well-fed and then set for a preliminary test to eliminate spontaneous lethal and sterility mutants. These tests were carried out for three days before treatment with the mutagens. Females laying few eggs or exhibiting low hatchability were eliminated from the experiments. All hatchability tests were carried out at 30 C, which is the optimum temperature for Habrobracon. Pans of water were placed in the incubators to provide a high humidity so that the dead eggs would not shrivel. Larvae will hatch normally at high or low humidity, but inviable embryos are difficult to score for time of death when they are even slightly shriveled. Virgin females were fed with fresh caterpillars of Ephestia, and for the night before treatment they were kept without caterpillars at 30 C. Oocytes in the late metaphase of the first meiotic division are stored in the uterine sac under these conditions (WHITING 1945). The following morning the animals were treated with an aerosol of nitrogen mustard or EMS. After treatment the animals were divided into two groups, The animals of one group were set unmated, each singly in a small stender dish with one fresh caterpillar. The other group was mated with No. 17 0% males, a stock with different sex alleles from No. 33, in a mass mating and then set singly. Both groups were allowed to lay eggs for six hours (A) on the first day. These eggs were all in metaphase I during treatment. After this period the animals were transferred into new dishes and kept without a caterpillar. The next morning each animal received a fresh caterpillar for a two-hour period (B). The eggs laid during this period were discarded because these represent a mixture of eggs in metaphase I and prophase I during exposure. After this short period the animals were transferred to new dishes and with new caterpil-

3 OOCYTE RESPONSE TO MUTAGENS 855 lars. The eggs laid then were in prophrjse I during treatment. The length of this period (C) was four hours. The wasps were again stored overnight without caterpillars until the following day when eggs were collected for a six-hour period (D)-these also were in prophase I during the treatment. Immediately after transferring the wasps to new dishes, the eggs were counted and, if displaced, were put back on the host caterpillar. Hatchability was recorded about 36 hours after removal of the wasps. At this time all surviving embryos had hatched and the stage of death of the dead embryos could be determined. From these data total embryo lethality can be estimated, and over 85 percent of these deaths are believed to be due to dominant embryo lethality. The larval and pupal mortalities were also scored but are not presented here since a recurrent disease killing the wasps preferentially during early pupal stages invalidated some of the data. To measure recessive embryo lethality, the surviving females from the mated groups of females were dissected from the cocoons as virgins. These were set singly, each with one caterpillar, for 12-hour periods (overnight without caterpillars) for three days. The hatchabilities were taken as described. The data were then summarized for each virgin. Those having a hatchability of 50 percent or less were counted as being heterozygous for a recessive lethal. In these cases death usually occurred at the same stage of embryogenesis in each dead egg. Nitrogen mustard (kindly supplied by Merck and Company) and EMS were used in these experiments. A time of treatment of 30 minutes was kept constant in all experiments, but the Concentration of the solution placed in the nebulizer was changed. A process diagram of the aerosol-generating apparatus is shown in Figure 1. The arrangement of parts of the apparatus was changed from the one previously described (VON BORSTEL 1955a). The air filter is merely a tube filled with glass wool. The rotameter was used to measure and observe the air flow during treatment. The air flow was kept constant and equal in all experiments. A five percent maximum flow was maintained for the aerosol, and a flow of 30 percent of maximum was maintained for the fresh air. This made it possible to B Air Ill 7 AEROSOL GENERATOR Ex haus Air Inlet Rotameter Alternating Valve System Condenser NaOH OT KOH in H@ or Alcohol Traps FIGURE 1.-Schematic diagram of the aerosol-generating apparatus. The solution being tested for its mutagenic effect is placed in the nebulizer and is transmitted as an aerosol to the wasps contained in the exposure chamber.

4 856 E. A. LOBBECKE AND R. c. VON BORSTEL repeat experiments with reasonable reliability. The alternation of air flow (10 seconds) and aerosol flow (50 seconds) was controlled mechanically by a synchronous electric motor. Other details were described previously ( VON BORSTEL 1955a). In order to compare the effects of mutagens on haploid eggs with the effects on diploid eggs, a method of estimating the number of diploid eggs in the mated group is necessary, since not every egg is fertilized. With this method, the amount of death in each stage attributable to unfertilized eggs can be estimated in the mated group. A method was developed by ATWOOD, VON BORSTEL and WHITING (1956) and later simplified (VON BORSTEL and REKEMEYER 1959) where the proportion of the fertilized eggs, f, is given by the equation where V, is the fraction of eggs that yield adults in the unmated group (males) and p is the ratio of surviving adult males to the total number of eggs from the mated group. In treated eggs, the frequencies of fertilized eggs dying at any stage of development, sf, were computed from the data in Tables 1 and 2 by where srn is the proportion of embryos from mated females that die at a particular stage of development and s, is the proportion of embryos from unmated females that die at the same developmental stage. RESULTS Uncorrected data for hatchability and for death during the different stages of embryogenesis after nitrogen mustard treatment are listed in Tables 1 and 2. In all tables the groups designated A represent treated metaphase I oocytes, and C and D, treated prophase I oocytes. Groups C and D are kept separate since oocytes in the D group were younger than oocytes in the C group when females were exposed to the aerosol, and thus constitute an oocyte class that might have a slightly different response to the mutagens. Each female was tested for hatchability before every experiment. The data for the females used in the experiments are listed as controls in the tables. Three different doses of nitrogen mustard were employed. It is clear from the data that oocytes in metaphase I are more sensitive than occytes in prophase I when lethality of offspring is the criterion. This had been shown previously with Habrobracon after exposure of virgin females to nitrogen mustard (VON BORSTEL 1955a). In Table 1 unmated and mated groups are compared in the case where no dominant lethality had been induced in the sperm by residual nitrogen mustard left on the bodies of the females or in the seminal receptacles. In other experiments, especially with higher doses, dominant lethality appeared to have been induced in the sperm by residual nitrogen mustard. The residual effect shows up as a

5 ~~ OOCYTE RESPONSE TO MUTAGENS 85 7 TABLE 1 Suruiual and stages of death of embryos after treatment of Habrobracon oocytes with nitrogen mustard Stages treated and concentration of nitrogen mustard Number of eggs Number of dylng animals at each stage of embryogenesis Adults Hatched Percent - eggs hatched Male Female One percent concentration Control jarmated Mated Unmated (Mated nmated tdu (Mated Two percent concentration Control *A Unmated tc Unmated td Unmated * Metaphase I. + Prophase I. higher incidence of lethality in the mated group than in the unmated group treated at the same time. These cases are therefore separately listed in Table 2. The effect is illustrated in Figure 2, which shows that fertilized eggs, especially in the C and D groups (estimated by equations 1 and 2), have a higher frequency of deaths in stage 1 of embryogenesis than do unfertilized eggs. At a concentration of one percent the effect is slight (Figure 2a), but with a concentration of ten percent the effect on the sperm is striking (Figure 2b). The residual effect of the chemical mutagen on sperm was always controlled internally in each experiment, since the same relative frequencies of stage 1 deaths were induced in the mated series of the C and D groups. The uncorrected data for hatchability and death during different stages of embryogenesis after EMS treatment are listed in Table 3. EMS is so toxic to the adult wasps that only two concentrations were used. Concentrations higher than 1.0 percent killed the wasps and concentrations lower than 0.5 percent did not induce dominant lethality. Hatchability curves for eggs from unmated females are shown in Figure 3. Data from group C only are used for the treated prophase I oocytes, since this is the group from which X-radiation prophase I data are drawn. In all cases, it is clear that the two chemical mutagens resemble X-radiation with respect to metaphase I having a higher sensitivity than prophase I. Even from these limited

6 858 E. A. LOBBECKE AND R. c. VON BORSTEL TABLE 2 Suruiual and stages of death of embryos aftel treatment of Habrobracon oocytes with nitrogen mustard where residual effect on sperm is indicated Number of dymg animals at Stages treated and concentration of Number Hatched Percent each stage of embryogenesis - Adults nitrogen mustard of eggs eggs hatched Male Female One percent concentration Control Mated Tmated Mated Mated Control Ten percent concentration Mated Tmated tdrmated Mated Mated ; Metaphase I 7 Prophase I a data, it would seem that the shape of the metaphase I curve for EMS is not exponential. Figure 4 shows the times of death during development for unfertilized and fertilized eggs after treating metaphase I and prophase I oocytes with nitrogen mustard. Of paramount interest is the shift in time of death from stage 1 in the unfertilized metaphase I eggs to later stages in the fertilized eggs. It can be seen by comparing the height of the bars that some of the fertilized eggs must have hatched that otherwise would have died in the unfertilized state. Significant data for this nuclear reactivation (VON BORSTEL 1961a) were obtained in one unrepeated experiment with nitrogen mustard; therefore, this phenomenon must be regarded as tentative and should be sought in further experiments. Times of death after EMS treatment are shown in Figure 5. Nuclear reactivation was not observed after fertilization. Quite the contrary, the corrected data for stage 1 deaths in eggs treated at metaphase I indicate a slight rise in lethality of the fertilized eggs. That this is not caused by dominant lethality being induced in the sperm is seen by examination of the C and D plots, where no signifiant rise in incidence of stage 1 depths of fertilized eggs is recorded. The higher le-

7 OOCYTE RESPONSE TO MUTAGENS b A C 0 - I Tl-l-l I STAGE OF EMBRYOGENESIS -.I - I FIGURE 2.-Frequencies 'oh death at different stages of development of embryos treated at metaphase I (A) and prophase I (C, D) of meiosis with one percent (a) and ten percent (b) solutions of nitrogen mustard. Solid columns, unfertilized; open columns, fertilized. Increased height of open columns in C and D indicated that sperm were affected by the residual chemical mutagen left on the females. thality of fertilized eggs in the metaphase group may be due to experimental variability, selective mating of the more heavily treated females, selective deaths of the more heavily treated females in the unmated group, or some other unknown factor. Table 4 shows the recessive lethal frequencies obtained after treatment with EMS, nitrogen mustard, and X-radiation. After treatment with EMS, metaphase I appears to be three times as sensitive as prophase I at the lowest exposure used, but the difference is not significant. After treatment with nitrogen mustard the two stages show a significantly different response for induced recessive lethal mutations. The two experiments at one dose for nitrogen mustard are listed separately since some of the data are taken from wasps where nitrogen mustard had induced some dominant lethality in the sperm. Apparently, however, no recessive lethals had been induced in the sperm (Table 4), but caution

8 860 E. A. LOBBECKE AND R. c. VON BORSTEL TABLE 3 Survival and stages of death of embryos after treatment of Habrobracon oocytes with EMS Number of dymg an~mals at each stage of embryogenesis Adults Stages treated and Number Hatched Percent doses of EMS of eggs eggs hatched Male Female ~ 0.5 percent concentration Control lunmated *A \Mated Di Unmated Mated Unmated Mated percent concentration Control Unmated *A[ Unmated Unmated Unmated Unmated cl Unmated ~ ~ * Metaphase I. f Prophase I e $(O' NITROGEN MUSTARD CONCN (%) I5 EMSCONCN (%) FIGURE 3.-Dose-action cuwes for Habrobracon metaphase I and prophase I oocytes treated with nitrogen mustard (a), EMS (b), and X-radiation (c). metaphase I; 0 prophase I. X- radiation data selected from Figure 2 in VON BORSTEL and REKEMEYER (1959).

9 OOCYTE RESPONSE TO MUTAGENS i a STAGE OF EMBRYOGENESIS FIGURE 4.-Frequencies of death at different stages of development of embryos treated at metaphase I (A) and prophase I (C, D) of meiosis with a one perceqt solution of nitrogen mustard. Solid L' columns, unfertilized; open columns, fertilized. A C D n +T-d4- i STAGE OF EMBRYOGENESIS FIGURE 5.-Frequencies of death at different stages of development of embryos treated at metaphase I (A) and prophase I (C, D) of meiosis with a 0.5 percent solution of EMS. Solid columns, unfertilized; open columns, fertilized. +l-l-b= must be exercised since it may be that the lack of a large sensitivity difference could be attributed to recessive lethals being induced in the sperm and these would account for the recessive lethality observed in the prophase I oocytes. This possibility is regarded as unlikely, however, since the EMS-treated females were mated one day after exposure to the mutagen and the females listed in Table 1 that were treated with nitrogen mustard had little, if any, dominant lethality induced in the sperm. The induction of recessive lethal mutations in the prophase I oocytes had not been expected to take place at the exposures used because the great difference in sensitivity for dominant lethality between metaphase I and prophase I is reflected also when recessive lethality is the criterion. The 20-fold difference between metaphase I and prophase I for X-radiation is reduced to a 3-to-5-fold difference for nitrogen mustard, and a zero-to-3-fold difference with EMS.

10 862 E. A. LOBBECKE AND R. c. VON BORSTEL TABLE 4 Recessive lethal frequencies after treatment of Habrobracon oocytes with mutagenic agents Mutagenic agent EMS Dose 0.5% 1.0% Metaphase Prophase - A c D A.C A:C+D 7/115= 6.1% 3/126= 2.4% 2/103=1.9% p= p=o.%o.i O/ 36= 0 I/ 47= 2.1% O/ 60=0 $pf=0.57 pf= % Nitrogen mustard *7/ 46=15.2% 3/ 88= 3.4% I/ 82~1.2% f2/ 49= 4.1% 0/ 91= 0 2/104=1.9% p= p<o.ool X-radiation$ (r) ,000 * F, females from Table I. + E; females from Table 2. $ From ATWooD et al. (1956). $ pf FISHER S exact test. 18/312= 5.8% 25/127=19.7% 21/138=15.2% 2/157= 1.3% 14/134=10.5% DISCUSSION The most striking feature of the data is that a large difference in sensitivity between metaphase I and prophase I exists for total embryo lethality with chemical mutagens and with X-radiation, but this difference is reduced considerably for the chemical mutagens alone when recessive lethal frequency is the criterion. The similar alkylating action for EMS and nitrogen mustard (BROOKES and LAWLEY 1961) and the relative lack of a differential stage sensitivity response for recessive lethals suggest that these chemicals, particularly EMS, induce mutations at a fundamental organizational level of the chromosome, possibly at the genic level itself. That X-radiation should act otherwise suggests an indirect role for this agent, indirect in the sense that its effects are subject to modification by metaphase and prophase stage differences in organization or chemistry. Though a straightforward postulate may be made about the induction of recessive lethals by chemical mutagens, the mechanisms by which alkylating agents induce dominant lethality are not evident at this time. If one compares the frequencies of death at every stage of development induced by X-radiation (VON BORSTEL and REKEMEYER 1959) with those induced by chemical mutagens, it can be seen that the type of lethality believed to be caused by inhibition of mitosis (VON BORSTEL 1960; VON BORSTEL and ATWOOD, unpublished results) is more frequent after chemical mutagen treatment and the types attributable to loss of chromosomes or their parts (VON BORSTEL and REKEMEYER 1959) are much less frequent. Lethality deriving from mitotic inhibition is restricted to developmental stage 1. It has been observed following ultraviolet irradiation that a large fraction of

11 OOCYTE RESPONSE TO MUTAGENS 863 this type of lethality is reversed by fertilization. This phenomenon has been called nuclear reactivation (VON BORSTEL 1961a). The nitrogen mustard appears to induce a similar reactivable nuclear damage, that is, death before blastoderm formation, with the damage at least partially reparable by a sperm nucleus. EMS also induces many of the embryos to die during very early development, but fertilization with normal sperm does not appear to reactivate the egg. It is thought that X-radiation-induced lethality brought on by inhibition of mitosis has as its underlying basis that of chromosome bridge formation (VON BORSTEL 1960; VON BORSTEL 1961b), but X-radiation also induces many deaths through deletions of chromosomes or their parts, something that EMS does not do. It is possible that EMS inhibits mitosis by some action other than by formation of chromosome bridges, as postulated for X-radiation, or by an action that cannot be reactivated, like ultraviolet radiation or nitrogen mustard. SUMMARY Ethyl methanesulfonate (EMS) and nitrogen mustard were used to induce dominant and recessive lethal mutations in the oocytes of the parasitic wasp Habrobracon. Oocytes in the first meiotic metaphase were found to be much more sensitive to EMS and nitrogen mustard (ca. 20 times) than oocytes in first meiotic prophase when dominant lethality was the criterion. In this respect, the action of the chemical mutagens resembles that of X-radiation. With recessive lethality as criterion, metaphase I and prophase I oocytes respond differently to X-radiation and chemical mutagens. A 20-fold difference prevails after X-radiation, with metaphase I being the more sensitive stage. With nitrogen mustard, metaphase I is three to five times more sensitive than prophase I, and with EMS, no significant difference was found between metaphase I and prophase I. ACKNOWLEDGMENTS We are grateful for the critical reading of the manuscript by DRS. F. J. DE SERRES, R. F. KIMBALL, D. R. KRIEG, and D. M. PRESCOTT, and for the statistical advice of DR. D. G. GOSSLEE. The first author wishes to acknowledge with gratitude the provision of laboratory facilities by DR. A. HOLLAENDER. and the financial support during the work by a stipendium from the Deutschen Forschungsgemeinschaft. LITERATURE CITED ATWOOD, K. C., R. C. VONBORSTEL, and A. R. WHITING, 1956 An influence of ploidy on the time of expression of dominant lethal mutations in Habrobracon. Genetics 41: BENZER, S., 1961 On the topography of the genetic fine structure. Proc. Natl. Acad. Sci. U. S. 47: BROOKES, P., and P. D. LAWLEY, 1960 The reaction of mustard gas with nucleic acids in vitro and in uiuo. Biochem. J. 77: The reaction of mono- and difunctional alkylating agents with nucleic acids. Biochem. J. 80:

12 864 E. A. LOBBECKE AND R. c. VON BORSTEL FAHMY, 0. G., 1961 Cytogenetic analysis of the action of carcinogens and tumour inhibitors in Drosophila melunogaster. XI. Mutagenic efficiency of the mesyloxy esters on sperm in relation to molecular structure. Genetics 46: FAHMY, 0. G., and M. J. FAHMY, 1961 Cytogenetic analysis of the action of carcinogens and tumor inhibitors in Drosophila melanogaster. X. The nature of the mutations induced by the mesyloxy esters in relation to molecular cross-linkage. Genetics 46: FREESE, E., 1961 The molecular mechanisms of mutations. Proc. Fifth Intern. Congr. Biochem., Moscow (in press). GOLDACRE, R. J., A. LOVELESS, and W. C. J. Ross, 1949 Mode of production of chromosome abnormalities by the nitrogen mustards-the possible role of cross-linking. Nature 163 : GREEN, D. M., and D. R. KRIEG, 1961 The delayed origin of mutants induced by exposure of extracellular phage T4 to ethyl methane sulfonate. Proc. Natl. Acad. Sci. U. S. 47: HEIDENTHAL, G., 1952 X-ray induced recessive lethals in Habrobracon. Genetics 37: 590. LAWLEY, P. D., 1957 The relative reactivities of deoxyribonucleotides and of the bases of DNA towards alkylating agents. Biochim. et Biophys. Acta 26: LAWLEY, P. D., and C. A. WALLICK, 1957 The action of alkylating agents on deoxyribonucleic acid and guanylic acid. Chem. & Ind. (London) : 633. LOVELESS, A., 1958 Increased rate of plaque-type and host-range mutation following treatment of bacteriophage in uitro with ethyl methanesulphonate. Nature 181 : The influence of radiomimetic substances on deoxyribonucleic acid synthesis and function studied in Escherichia coli phage systems Mutation of T2 bacteriophage as a consequence of alkylation in uitro: the uniqueness of ethylation. Proc. Roy. Soc. London B 150: The involvement of deoxyribonucleic acids in chemical mutagenesis. Biochem. Pharmacol. 4: STRAUSS, BERNARD S., 1961 Specificity of the mutagenic action of the alkylating agents. Nature 191: VON BORSTEL, R. C., 1955a Differential response of meiotic stages in Habrobracon eggs to nitrogen mustard. Genetics 40: b Feulgen negative nuclear division in Habrobracon eggs after lethal exposure to X rays or nitrogen mustard. Nature 175: Sulla natura della letaliti dominante indotta dalle radiazioni. Atti Assoc. Genet. Ital. 5: a Induction of nuclear damage by ionizing and ultraviolet radiation. pp Prog. in Photobiol., Proc. 3rd Intern. Congr. Photobiol., Copenhagen, Edited by B. CHR. CHRISTENSEN and B. BUCHMANN. Elsevier Publishing Co. Amsterdam, Netherlands. 1961b The genetic basis for X-radiation-induced mitotic inhibition. Radiation Research 14: 453. VON BORSTEL, R. C., and M. L. REKEMEYER, 1959 Radiation-induced and genetically contrived dominant lethality in Habrobracon and Drosophila. Genetics 44: WESTERGAARD, M., 1957 entia 13: Chemical mutagenesis in relation to the concept of the gene. Experi- WHITING, A. R., Effects of X-rays on hatchability and on chromosomes of Habrobracon eggs treated in first meiotic prophase and metaphase. Am. Naturalist 79: WHITING, A. R., and W. E. MURPHY, 1956 Differences in response of irradiated eggs and spermatozoa of Habrobracon to anoxia. J. Genet. 54: