CYTQLOGICAL AND! GENETEC QESERVATEQNS @F MEIQTEC @RWE EN @ROSQPHELA MELANGGASTEE Timer: €00 Hm Degree :2“ Mt. D. MiCHEGAN SYATE UNIVERSHY Mary Fae Range 3%? ‘ ._._-__, 4_ “A“, 0‘. LIBRARY L’ i*.-'lit‘higan Stats '3 University '7‘! 8812 13;“ i i S By This is to certify that the thesis entitled Cytological and Genetic Observations of Meiotic Drive in DROSOPHILA MELANOGAS‘I'ER presented by Mary Fae Rengo has been accepted towards fulfillment of the requirements for Ph.D. Zoology degree in C::;2:ou7;g Jz'fiiééncoéva A - F . Yande IMajor professor Date May 15, 1969 0-169 1W ‘ 1 MI an ill] III I 312 xix/II / HIM/Mil W U '69 .493“ i I ‘ G I. . -- s L 4 3 <:> Mary Fae Rengo 1969 ALL RIGHTS RESERVED ABSTRACT CYTOLOGICAL AND GENETIC OBSERVATIONS OF MEIOTIC DRIVE IN DROSOPHILA MELANOGASTER BY Mary Fae Rengo Many exceptions to Mendel's law of independent segre— gation have been noted. If the inequality of recovery of two homologous chromosomes is due to a meiotic event, these homologues are said to exhibit meiotic drive. This study was designed l) to ascertain the effect of irradiation at the onset of meiosis on the meiotic mutant Segregation-distorter in order to determine the meiotic stage during which the mechanism of §2_is sensitive, and 2) to observe the meiotic drive systems of §annd the abbreviated sc4sc8 X together ("Double—Drive") both cytologically and genetically at 18° and 28°C. Analysis of the interaction of these systems and correlation of the nonrandom polar segregation of sc4sc8 with its genetic recovery tested the hypothesis that the mechanism of both drive systems involves a predetermined functional-nonfunctional polarity which is independent of the drive systems which respond to it. Irradiation treatments were administered to very late third instar male g9 larvae. To observe the effects of Mary Fae Rengo sperm storage, part of the experimental and control groups were aged after eclosion as virgins. The first four daily broods from unstored irradiated males exhibited depressed 5_values. The percentage of aberrations and the depression of the §_values per brood appear to be positively correlated. Using the proportion of aberration products as an index, stored males appear to transfer few sperm which were in meiotic or premeiotic stages at the time of treatment. Depressed 5_values were exhibited by both irradiated and control groups which were stored. Lack of mating activity and possibly resultant prolongation of Meiosis I appear to "unstabilize” and depress the drive of §Q. The sex ratios (Ra/total) of g§_bw_progeny in experi- mental and control groups were in excess of 60%m A modifi- cation of Hiraizumi's §er chromosome homology hypothesis is presented to account for the lack of a reciprocal decrease in the sex ratios of §Q progeny. In the study of "Double-Drive,” the nonrandom segre- gation of sc4sc8 or the nondisjunctional nullo product away from the bacterial pole correlated with genetic recovery, supporting the hypothesis that a predetermined functional- nonfunctional polarity exists in the primary spermatocyte. At 18°C, sc4sc8 expresses increased drive when in the presence of s2, Both drives are slightly reduced when to- gether at 28°C. Thus, the data indicate that the drive 4sc8 systems of §2 and sc are not competitive in their inter- action, and that the mechanism of §Q is not independent of that to which sc4sc8 responds. CYTOLOGICAL AND GENETIC OBSERVATIONS OF MEIOTIC DRIVE IN DROSOPHILA MELANOGASTER BY Mary Fae Rengo A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1969 '0 El N be \\) \\) 1“ a) \. __.-I To my mother and father ii ACKNOWLEDGEMENTS I express my sincere thanks to Dr. Armon F. Yanders for his capable guidance and sincere interest and encourage- ment during the course of my graduate work. I acknowledge and thank the members of my guidance committee: Dr. G. B. Wilson, Dr. J. R. Shaver, Dr. J. E. Trosko, and Dr. J. V. Higgins. The sad loss of Dr. Wilson from this committee was sorely felt. The counsel and assistance he freely extended to me proved to be an invaluable part of my graduate education. I am grateful to Mrs. B. R. Henderson both for her generous help and advice, and for her warm interest in my work. This study was conducted during the tenure of a National Institutes of Haalth Fellowship and partially supported by a grant to Dr. A. F. Yanders from the U.S. Atomic Energy Commission (Contract AT(ll-l)-1033). iii TABLE OF CONTENTS Chapter Page I O IMRODUCTION O O O O O O O O O O O O O O O O 1 Effect of Irradiation 6 ”Double-Drive" 6 Genetic Tests 7 Cytological Tests 8 II C MTERIALS Am MTHODS O O O O O O O O O O O l O Irradiation of §2_ 10 Cytological and Genetic Studies of Meiotic Drive 11 Genetic Tests of Male Sibs of Dissected Males 13 Stocks 14 Derivation of ”Stock A“ with Granules 15 III 0 RESULTS 0 O O O O O O O O O O O O O O O O 0 16 Effect of Irradiation on §Q_ 4 l6 Cytological Observations of ac sc8 and "Double-Drivi" 21 Genetic Tests of SC SC8 and ”Double-Drive" 31 Correlation of Cytological Studies and Genetic Evidence 36 IV. DISCUSSION 0 O O O O O O O O O O O O O O O O 42 The Effect of Irradiation on §2_ 42 Relationship of Cytological and Genetic Data 45 V. SUMRY C O O O O O O O O O O O O O O O O O 51 BIBL IWMPHY O O O O O O O O O O O O O O O O O O O O O 5 3 APPEme O O O O O O O O O O O O O O O 0 O O O O O O O 57 iv LIST OF TABLES Table Page 1. SD-72 third instar larvae irradiated (450 r). unstored, control k = 95.8, 50 males, Experiment 1 . . . . . . . . . . . . . . . l7 2. 20 control males, unstored, Experiment 1, no aberrations Observed . . . . . . . . . 17 3. SD-72 third instar larvae irradiated (450 r), unstored, control k = 99.5, 25 males, Experiment 2 . . . . . . . . . . . . . . . l8 4. 10 control males, unstored, Experiment 2, no aberrations observed . . . . . . . . . 18 5. 15 SD—72 third instar larvae, irradiated (450 r), stored 7 days, replicate 2 . . . 19 6. 10 SD-72 third instar larvae, control group, stored 7 days, replicate 2 . . . . . . . . 19 7° Cytological observations . . . . . . . . . . . 28 8. Summary of cytological observations . . . . . 29 9. Summary of progeny tests - "Double-Drive" . . 32 10. “Double-Drive" segregation frequencies . . . . 33 11. Summary of progeny tests sc4sc8,y . . . . . . 33 12. Correlation of cytological and genetic observations . . . . . . . . . . . . . . . 37 13. Cytological observations, genetic observations 38 14. Test for homogeneity of means . . . . . . . . 39 LIST OF FIGURES Figure Page 1. k_values per daily brood of irradiated and control males, Experiment 1 . . . . . 23 2. k_values per daily brood of irradiated and control males, Experiment 2 . . . . . . . 25 3. Percent aberrations per daily brood of stored and unstored irradiated males, Experiments 1 and 2 . . . . . . . . . . . 27 vi I . INTRODUCTION Many exceptions to Mendel's law of independent segregation have been noted. These abnormal progeny ratios may be due to l) aberrant meiotic segregation, 2) gametic selection, or 3) post—fertilization selection. If the in— equality of recovery of two homologous chromosomes is due to a meiotic event, these homologues are said to exhibit meiotic drive (Nbvitski and Sandler, 1956). One of the meiotic drive phenomena occurring in Drosophila melanogaster, Segregation-distorter (SQ), has been intensively studied. §Q_is known to exhibit the follow— ing properties: 1) A male heterozygous for §Q_and a structur- ally normal §Q_chromosome produces almost all SQ progeny, with rare exceptions. 2) Females heterozygous for go produce normal segregation ratios; their heterozygous sons exhibit abnormal k values (with certain exceptions). (The parameter k_is defined as the proportion of functional SQ: bearing Sperm.) 3) When the §2_chromosome is heterozygous with a structurally rearranged homologue having one break- point near the centromere, such as ln(g§§)gy, segregation- distortion is inhibited. 4) An §Q_chromosome appears to be insensitive to the action of another §Q_chromosome. 5) The complex SQ locus lies in the centromeric region of chromosome II, between purple and Cinnabar. To the right of SD is l Activator of §Q,Ag(§2), which is necessary for §Q_to exhibit distortion. 6) There is a stabilizing modifier of §QL§§(§Q), at, or near, the tip of IIR. (Sandler, Hiraizumi, and Sandler, 1959; Sandler and Hiraizumi, 1960a, b, 1961a: Dennell and Judd, 1968). Sandler, Hiraizumi, and Sandler (1959) presented a model for SD action which assumed that SQ caused a break at SDI. The lethality of the union of §Qf sister chromatids would cause an excessive recovery of §erearing chromosomes. Their model was supported by preliminary cytological obser- vations of dicentrics at Metaphase II and bridges at Anaphase II in about half of the division figures examined (Crow, Thomas, and Sandler, 1962). Peacock and Erickson (1965) did an extensive cyto- logical investigation of meiosis in the SQ male. Completely normal spermatogenesis was found. They found that all pro- ducts of meiosis in the male are cytologically evident and are presumably transferred to the female. A Drosophila melanogaster male can transfer 3,000 to 4,000 sperm to the vagina during a single mating (Kaufmann and Demerec, 1942). The capacity of the female storage organs is about 700 sperm (Kaplan, Tinderholt, and Gugler 1962: Lefevere and Jensson, 1962). Peacock and Erickson reported that, when the number of sperm transferred by §Q_males was less than the storage capacity of the female the ratio of stored Sperm to progeny was 2:1. This ratio approached 1:1 when the number of sperm transferred exceeded the storage capacity of the female or when the insemination was of the order of 60 sperm or less. These same relationships were exhibited by control, wild—type males. In order to explain abnormal ratios recovered from certain translocation males 3 (1:4).83, Novitski and I. Sandler (1956a, b) proposed that some of the sperm produced by a Drosophila melanogaster male were regularly nonfunctional, and that particular chromosomes had certain propensities for being included in functional sperm. The cytological observations and the Sperm count versus progeny count ratios reported by Peacock and Erickson support this functionality hypothesis, and indicate that functional and nonfunctional Sperm are a regular aspect of spermiogenesis in Drosophila. Peacock and Erickson postu- lated that the mechanism of Segregation-distorter was non- random orientation of the SQ chromosome to the functional pole at Maiosis I. The propensity of SE to orient towards the proposed functional pole of Meiosis I appears to vary. §Qrbearing chromosomes with different strengths (meiotic drives) have been found in many natural populations, and exhibit k values from about .7 - 1.0 (Mange, 1961: Greenberg, 1962: Hiraizumi and Nakazima, 1965). At least some §Q_chromosomes are temperature sensitive. From treatment of four different §Q chromosomes with heat and cold, Mange (1968) found that temperature sensitivity was dependent on the stage of meiosis which a sperm is undergoing and independent of the age of the male being treated. The most sensitive period occurred around early meiosis. The §2_chromosomes which She reported to be greatly sensitive to heat treatment (30°C.) were recombinants. Both recombinants and natural §2_chromosomes exhibited vary- ing sensitivity to cold (190 C.). Other meiotic drive systems in Drosophila melanogaster have been shown to be temperature sensitive at early meiosis, with the effect of temperature treatment being a more normal segregation ratio. The §Q_effect (high recovery rate of the X chromosome) is greatly depressed at 18°C, with the sex ratio (SQ/total) being most reduced 7-8 days after treat- ment (Erickson and Hanks, 1961). Segregation ratios of both XD: IV and XP: Y from A-type Bag of gtgge translocation males were very much altered by 18°C treatment of prepupae (Zimmering and Perlman, 1962). Gershenson (1933) suggested that the unequal recovery of X0 and XXY progeny resulting from primary non-disjunction in the Drosophila male was due to chromosomal loss because of lack of synapsis. In XY and XYY males with chromosomes which differed in amount and distribution of heterochromatin, chromosome loss appeared to occur only where homology was drastically reduced, supporting Gershenson's hypothesis that lack of pairing is related to apparent loss of chromosomes (Sandler and Braver, 1954). Yet, males with sex chromosomes with greatly reduced homology exhibit more normal recovery of the Y chromosome at 180C than at 260C (Zimmering, 1963). Since a modified univalent Y shows a similar increase in recovery at 18°C, the increase in recovery of the Y from XY males observed at cold temperatures does not appear to be due to increased pairing between X and Y. Using sc4sc8/Y/Y males, Zimmering and Green (1965) found that the frequency of XY Sperm re- covered increased from 23% at 26°C to 43% at 18°C. Since, in such males, the sc4sc8X is usually a univalent while the Y chromosomes form a bivalent, the normalizing effect on segregation again does not appear to be due to increased pairing. Hewever, this was not demonstrated cytologically. Peacock (1965) found that, while failure of the sc4sc8X and Y chromosomes to synapse was common, these sub- sequent univalents usually travelled to the same pole at Anaphase I. The nondisjunction frequency was directly re- lated to the frequency of synaptic failure. The occurrence of meiotic loss of unpaired chromosomes was negligible. Peacock postulated that the SC4SC8 meiotic drive system was another case of nonrandom segregation of both disjunctional and nondisjunctional reciprocal products into functional and nonfunctional Sperm. The experiments in this study were undertaken to 1) ascertain the effect of irradiation at the onset of meiosis on Segregation-distorter and 2) observe two meiotic drive systems together, the Sc4sc8X and S2 ("Double-Drive”), both cytologically and genetically, at 180 and 28°C. Effect of Irradiation The effects of heat and cold Shock on §Q_and other chromosomes which exhibit abnormal segregation implicate early meiosis as the time of operation of these drive systems. Temperature treatments, however, are administered over rela- tively long periods of time. As a result, it is impossible to determine precisely the meiotic stage (or stages) which is critical to the drive systems. The functionality hypothe- sis would require the sensitivity to treatment to be exhibited prior to Anaphase I. Irradiation treatments can be administered over periods of very brief duration. By irradiation of very late third instar larvae, primary spermatocytes will be the most advanced meiotic stage undergoing treatment (Cooper, 1950). Sensitivity of §Q_at this stage would support the hypothesis that functional and nonfunctional Sperm are determined a Meiosis I and that the operation of the Segregation-distorter chromosome involves nonrandom orientation to the functional pole. "Double-Drive” The proposed functional and nonfunctional poles at Meiosis I provide a good working hypothesis to explain the mechanism of all meiotic drive phenomena. The operation of the meiotic drive systems of SQ and sc4sc8 has been attributed to the nonrandom segregation of the chromosome or chromosomes involved to the functional or nonfunctional pole at Anaphase I (Peacock, 1965; Peacock and Erickson, 1965). Analysis of the interaction of these systems together would test the hypothesis that the operation of both systems involves a predetermined functional-nonfunctional polarity which is independent of the drive system or systems being studied, or, if functionality is not predetermined, would provide information concerning the time of determination of the functional pole. Genetic Tests If the polarity of the primary Spermatocyte is determined prior to Anaphase I, the two systems would not be competitive. If both phenomena utilize functional-non- functional polarity, the drives exhibited by §Q_and the sc4sc8 systems in ”Double-Drive" should be Similar to those exhibited by these chromosomes when alone. In addition to the factors mentioned above, there are possible segregational interactions between the chromosomes involved. This would be reflected in further deviation from randomness in the association of chromosomes in recovered sperm. If the driving chromosomes themselves determine the functional pole, sc4sc8 and §Q would be in a competitive situation if the mechanisms of both systems involved functionality. When these systems are present in the same role, there are eight possible gametic products: Disjunctional products NOndiSjunctional products s9 sc4sc8 ‘SQ sc4sc8 Y §Q_Y §Q_nullo so+ sc4sc8 SD+ Sc4sc8 Y SD+ Y SD+ nullo If SD is stronger than sc4sc8 in polar determination, the drive of §Q in the "Double-Drive" Situation would approxi- mate the drive it exhibits alone, whereas the drive of the disjunctional and nondisjunctional products of the sc4sc8 system would be reduced due to the loss of sc4sc8 products which resulted in nonfunctional gametes. The reverse would be true if §Q_is the weaker competitor. If both systems are equal in polar determination strength, a high proportion of §Q_sc4sc8 and SD nullo products would be recovered, due to the competition between SD+ Y and SQ sc4sc8 and between §Q nullo and so+ sc4sc8 Y. Cytological Tests The abbreviated sc4sc8 chromosome can be distinguished from a normal X and from its Y homologue, thus allowing cyto- logical observation of its segregational behavior at Anaphase I. This behavior was scored in the sc4sc8 X System, and with the sc4sc8 X system in the presence of s2 ("Double-Drive”) at 180 and 28°C. The granular inclusions reported by Peacock and Erickson (1964) were present in the stocks used in this study. These granules have been shown to be Bacteria or Rickettsiae, and their polar association was concluded to be an independent response to the cytoplasmic gradient in meiotic cells to which ”driving” chromosomes respond (Yanders, Brewen, Peacock, and Goodchild, 1968). The presence and position of these inclusions in relation to the segregation of the chromosomes were noted. A positive correlation between non-random segregation to the pole lacking granular inclusions and subsequent genetic re- covery would support the conclusion of Yanders §t_al_(l968) that the polar association of these inclusions reflects a cytoplasmic gradient in meiotic cells associated with the determination of functionality of the gamete. With the polar association of the granules, the segregational behavior of sc4sc8 alone and in the presence of S2 can be examined. If the granules are indicators of polarity, a difference in the behavior of sc4sc8 in the two systems should be supported by genetic recovery ratios of the reciprocal products. Evidence will be presented which supports the hypo— thesis that the mechanisms of the §2 and sc4sc8 meiotic drive systems involve nonrandom segregation to pre-determined functional and nonfunctional poles at Anaphase I. The data also indicate an interaction between the two systems and apparent repulsion between the §erearing chromosome and the sex chromosomes which is influenced by temperature. II. MATERIALS AND METHODS The stocks of Drosophila melanogaster used are listed below. Nutrient medium, modified after Carpenter (1950), was used for all experiments. All virgin females used were aged four to five days before mating. Irradiation of SD Very late third instar nglg male larvae were irradi- ated with 0 (Control) or 450r of X-rays with a General Electric Maximar—250-III, operating at 250kv, 15ma, with a .5mm copper, 1.0mm aluminum filter, giving an average dose of llOr/minute. The larvae used were from half-hour egg collections. For treatment, control and experimental larvae were suspended in 0.7% saline solution (Beadle and Ephrussi, 1936), .5mm in depth, in an open plastic petri dish. In experiment I, 20 control and 50 experimental males were transferred without etherization to two virgin g§_§w females per day for seven days from the day of eclosion. In experiment II, 10 control and 25 experimental males were transferred as described above for seven days, from the day of eclosion. Ten control and 15 irradiated males were stored for seven days as virgins, then mated as above for seven days. All progeny resulting from these matings were collected and scored for sex and eye color. Male progeny 10 11 with aberrant colors were tested for meiotic drive by matings with cn bw virgin females. Cytological_and Genetic Studies of Meiotic Drive The sc4sc8 X chromosome used was a crossover product derived from an In(l)sc4, In(1)sc8 female. The distal break- points of these two inversions are similar. The proximal breakpoints, in the basal heterochromatin, are such that the crossover chromosome Ins(l)sc4sc8, having the distal region of In(1)sc4 and the proximal region of In(l)sc8, is deficient for a large portion of basal heterochromatin, (Cooper, 1959). 4 8 + . . For the study of the Ins(l)sc Sc ,y,y Y meiotic drive system, alone and in the presence of SD, the sc4sc8 stock, stock A, was infected with granules from g§_by_ females known to exhibit cytoplasmic granules. The granules are maternally inherited. Crosses between sc4sc8,y/y+Y 83’ and sc4sc8, yzBasc $2 (with granules) or between ++Zy+Y: SD, cn bw/SD+++ d8’(Stock B) and sc4sc8, y/Basc $9 (with granules) yield two types of F1 . 4 8 + + able cytologically: sc sc , y/y Y and Basc/y Y. males which are distinguish- 1. _s_c4$c8 without §Q sc4sc8Ly; cn bw 29 x sc4sc8,y: cn bw d9! Basc cn bw y+ Y cn bw sc4sc8,y: cn bw sc4sc81y: cn bw Sc4sc8,y; cn bw Basc: cn bw Basc cn bw Basc cn bw y+ Y cn bw y+ Y cn bw Cytologically distinguishable males 12 2. sc4sc8 with S2 ("Double-Drive") sc4sc8,y; cn bw 29- x + 7 SD (C! + Basc cn bw y Y en bw sc4sc8,y7 cn bw Basc: cn bw sc4sc8,y: cn bw Basc; cn bw + on bw + cn bw y+ Y cn bw y+ Y cn bw 4 8 4 8 sc sc ,y7 cn bw Basc: cn bw sc sc ,y; en bw Basc; cn bw + so + so y+ Y so y"' Y cn bw Cytologically distinguishable males The sib males with Bag; X served as controls. These crosses were cultured at 180 and 28°C. Testes from F1 prepupae and very early pupae were dissected in 0.7% saline, stained in acetoorcein, then squashed for observation under phase contrast. The prepupal and early pupal stages were selected because granule populations diminish greatly after these stages. All primary spermatocytes with suitable Anaphase I divisions were scored. These cells were divided into three categories of granule populations: 1) polarized, where the granular mass is concentrated at one pole, 2) non—polarized, where the granular mass is distributed throughout the cell, situated between the poles at the equator, or evenly distri- buted at both poles, and 3) those cells with few or no granules. Disjunctional and nondisjunctional divisions were scored. In the case of polarized cells, the chromosomes travelling to the granular and non-granular poles were noted. 13 The Sibs of those males dissected were mated to y} cg by females for genetic tests of the sc4sc8 drives, nondisjunction rates and k_(SD/total) values of the F2 progeny. These crosses were cultured at 22.50C. Genetic Tests of Male Sibs of Dissected Males l. sc4sc8 without SQ .‘U gr1__b_w_ 99* x sc4sc8.y: cnbw 6’! Y cn bw y+ Y en bw sc4sc8,y: cn bw y ; cn bw nullo; cn bw .SC4SC8:Y7 cn bw y cn bw y+Y cn bw y cn bw y+ Y cn bw / y from disjunctional gametes from nondisjunctional gametes 2. sc4sc8 with §2_ y: M 99?- x sc4sc8,y: cn bw 6'0] y cn bw y+ Y SD sc4sc8,y: cn bw y 7 cn bw nullo: cn bw sc4sc8,y: cn bw y cn bw y+Y cn bw y en bw y+ Y cn bw Y sc4sc8; cn bw y : cn bw nullo: cn bw sc4sc8,y; cn bw y so y+Y so y so 2+ Y so Y / from disjunctional gametes from nondisjunctional gametes 14 Stocks SD Segregation-distorter, SD-72, second chromosome (Sandler, Hiraizumi, and I, Sandler, 1959). cm bw Cinnabar and brown, second chromosome, exhibits granules (Madison). y,: cn bw yellow, Cinnabar, and brown (derived by Grant Brewen yp-32 (Oregon) and cn bw, Madison). Stock B: ; cn bw 32 x + ; SD-72 dé’(derived by Yanders). + + cn bw y+ Y cn bw Males used for ”Double-Drive" cross. "Stock A with granules": Basc ; cn bw 23 x sc4sc8,y; cn bw o’c! sc4sc8,y cn bw y+ Y cn bw Females used for "Double-Drive” cross. ”Stock A with granules" derived from: 1. Yanders' "Stock A": sc4sc8,y: cn bw 99 x sc4sc8fi; cn bw Jo] Clb cn bw Y en bw 2. Base (Philadelphia). 3. + 3 en bw dé’from "Stock B". + y Y 4. cn bw with granules, (Madison). 15 Derivation of "Stock A with Granules" 1. Basc 7 + + 33 x 7 en bw 69’ + Basc + +-’////, ‘Y cn bw 2. Base ; cn bw x .i ; cn bw 22 Y + + + cn bw (granules) 3. Basc ; cn bw x sc4sc8,y: cn bw d6, + on bw Y cn bw (granules) Basc : cn bw 29 4 8 so sc ,y: cn bw (for "Stock A" with granules) 4. sc4sc8,y; cn bw 39 x + : cn bw ClB cn bw Yy+ cn bw sc4sc8Ly; cn bw dé/ Y'y+ cn bw (for "Stock A" with granules) III. RESULTS Effects of Irradiation on SD Tables 1 through 6 and Figures 1 through 4 summarize the results of irradiating SQ male larvae at or near the on- set of meiosis. In experiment 1, the k_value (SQ/total) of the progeny of irradiated unstored males was depressed below that of the control group for the first Six days of mating, with the lowest k_values resulting from days 2 and 3. The brood of day 7 exhibited a k_higher than any of the control group. In experiment 2, the k_values of the un— stored, irradiated males were depressed in all seven daily broods, with day 4 showing the greatest depression. Due to the large numbers of progeny, a test of Significance is not necessary. Certain aberrations appeared in progeny of irradiated larvae which did not occur in control groups. These aber- rations provide an indication of which sperm batches were most affected by irradiation. The aberrations detectable are Cinnabar and brown eyed flies. These aberrant progeny are probably the result of radiation-induced chromatid breaks between the Cinnabar and brgwg loci. Breaks in analogous regions of non—sister chromatids and subsequent chromatid exchange would produce such aberrants. The §Q_Ac SD lgg+ chromatid would thereby carry the bw_allele but no stabilizer l6 17 Table l. SD-72 third instar larvae irradiated (450 r), unstored, control k = 95.8, 50 males, Experiment 1. Paternal Age k(SD/total) Percent Total Total in Days Aberrations Progeny Aberations bw on 1 92.96 .47 428 2 0 2 85.72 .63 1265 4 4 3 85.62 .24 3701 8 l 4 91.79 .11 5534 6 0 5 92.31 .08 6155 4 1 6 90.32 .07 6120 3 0 7 97.25 .02 6120 l 0 Table 2. 20 control males, unstored, Experiment 1. No aberrations observed. Paternal Age k Total In Days Progeny l 95.71 632 2 95.69 1974 3 95.83 3302 4 95.71 2571 5 95.87 2699 6 96.01 2878 7 95.81 3005 18 Table 3. SD-72 third instar larvae irradiated (450 r), unstored, control k = 99.5, 25 males, Experiment 2. Paternal Age k(SD/total) Percent Total Total in Days Aberrations Progeny Aberrations bw cn l 97.84 .37 1067 4 0 2 97.95 .42 2405 10 0 3 96.44 .22 2249 5 0 4 95.98 .49 2436 10 0 5 96.82 .09 3430 3 0 6 97.02 .00 3695 0 0 7 97.09 .025 3954 1 0 Table 4. 10 control males, unstored, Experiment 2. No aberrations observed. Paternal Age k Total In Days Progeny 1 99.43 1051 2 99.20 1496 3 99.44 1070 4 99.46 1303 5 99.36 776 6 99.64 822 7 99.42 517 19 Table 5. 15 SD-72 third instar larvae, irradiated (450 r), stored 7 days, Replicate 2. Paternal Age k Percent Total Total In Days Aberrations Progeny Aberrations .10.! so. 7 97.86 .089 3360 3 0 8 97.11 .000 2389 0 0 9 98.41 .000 2008 0 0 10 98.68 .000 2039 0 0 11 97.66 .000 1880 0 0 12 97.09 .000 1753 0 0 13 98.61 .000 2083 0 0 Table 6. 10 SD-72 third instar larvae, control group, stored 7 days, Replicate 2. Paternal Age k Percent Total Total in Days Aberrations Progeny Aberrations 7 99.23 none 2691 none 8 98.22 2139 9 96.77 1918 10 96.48 1703 11 99.33 1649 12 97.99 1541 13 98.45 1160 20 locus, St(SD). As such, it would exhibit a moderate, un- stable 3, The SD+AC(SD)+cn chromatid would bear the normal allele for brown, bw+, and the £12. stabilizer, Stgso), and exhibit no drive. All gg_and bw_male progeny were tested for drive by mating them with g§_by virgin females. The mean k_for b£9w§_ males was .772. The k_for cinnabar males was .521. Irradiated males mated from day of eclosion Show the greatest number of aberrations in broods from days one through four in both replicates 1 and 2. The percentage of aberrations and the depression of k values per brood appear to be positively correlated. Due to the small numbers of aberrations in the data concerned, a nonparametric test of correlation is not applicable. Aberrations appeared only in the first brood of the stored, irradiated males. These stored males exhibited k values similar to that of the stored control group. Hew- ever, lower k_va1ues were exhibited by the stored control group than those of the unstored males. Lack of mating activity appears to increase the percentage of SQ: sperm transferred to the female. If aberrations are an indication of which Sperm batches were in meiotic or premeiotic stages at the time of treatment, the stored males do not appear to transfer most of these sperm. The over-all sex ratio (QR/total) of EE.§E progeny from experiment 1 was .619. This represents a total of 2484 .gg_bw progeny. Experiment 2 gn.§w progeny (682) had an 21 over-all sex ratio of .625. This supports the hypothesis of Hiraizumi and Nakajimi (1967) that §2_has some homology with the X chromosome which reduces the probability of both travelling to the same pole. Where the SQ: 9g.by_chromosome reached the functional pole, it was accompanied by the X chromosome over 60 per— cent of the time. Thus, when S2 2g: bw:_travelled to the hypothesized non-functional pole at Anaphase I, it was accompanied by the Y chromosome more than 60 percent of the time. This implies a degree of repulsion between the §Qrbearing chromosome II and the X chromosome comparable to the repulsion exhibited between homologues. 4 8 Cytological Observations of Sc sc and "Double-Drive” The results of the cytological studies are given in Tables 7 and 8. The presence and position of the granular inclusions varied. In about half of the primary spermato- cytes of all classes observed, the granules were polarized, the great majority of granules being situated in a mass or near one pole of the cell. The Basg control males exhibited polarization to about the same extent as the experimental groups. The granular masses of nonpolarized cells in both types of males were usually distributed on one side of the cell along the equator. The position of the granular inclusions was undoubted- ly disturbed by the squash procedure. In most cases where the granules were scored as polarized, the granular mass was 22 Figure l. k_values per daily brood. Irradiated and control males, Experiment 1. values 100 1 23 96 -4 . ____ O—___.__.——O——--.‘\‘ 92 - O O 88 - ./ O 84- 78‘ —u——- control males irradiated males I l l l r l l 2 3 4 5 . 6 Paternal Age in Days 24 Figure 2. k_va1ues per daily brood. Irradiated and control males. Experiment 2. values k 25 -- Unstored control males -———— ‘Unstored irradiated males -—-- Stored irradiated males __.__. Stored control males 1001K /\ /\ \ k‘fi \ / \ \V \\ / \ /’.\\ // \ \/ \ /’-—*‘~v’ N \ O . \‘O/ . \ 99— \ /\ \\\/// \ x". / \ r \ I \J \ /, 981/. \ I/ l\ \/' \\ x! \s I, \‘ '\ / \s\ ,I 97.1 '/. \ / l ‘ I s , I ./ \l \\ I \‘.l . / ° \- 96 I l 7 I I l l l l l l l I I I I l 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Paternal Age in Days 26 Figure 3. Percent aberrations per daily brood. Irradiated males, Experiments 1 and 2. 27 .60 T .55 - -—-——- Unstored irradiated males, Experiment 1 «————— Unstored irradiated males, -50 - - Experiment 2 ---“- Stored irradiated males, .45 q Experiment 2 .40 - m c 3 35 u ' ' m i: B .30 - n: 2 .25 . w u H m O4 020 " .15 - 010 -‘ 005 -' . \ \ . \ O \ \ /\: ‘ L W l IiT F I l T Figure 3. 12345678910111213 Paternal Age in Days Percent aberrations per daily brood. Irradiated males, Experiments 1 and 2. 28 =w>HHQImanson:««* HMCOHuchanQ*¥ HmcoHuUGSHchcoz« mmma mma mm m¢ mm ¢¢ ommm mad m as ma Hm m m me am mus oma mma ma am me He ommm mmH NH om m an ma mm me am .*..o.o omH mam ma mm me he ommm mom mm Hm pa mm as mm «m I ma mus omm «mm am do we do ommm cam mm ma mm me am am ::&n, mm *««.o.n 0mm moan a. a... 3 a. 3mm 3 am. m. ”MM umwm am.“ .538 o .92 ¥.Q.Z ¥¥.nmfln «.Q.Z *¥.hmfla *.Q.Z Hmcow#UG5anQ .mGOHum>Hmmno HMUHmOHOp>U .n magma 29 was. 08. m.mm new 33 m.mm m.¢m mma. mmm. ¢.oo mumfiom m.mm 000. «.mm Dam ommm H.mm m.om mmm. wmm. m.ao m>flhnloansoa .Uoma >.m¢ ooo. m.mm me ommm N.¢m H.¢m fimv. mmm. m.mv womfiom o.mm ooo. N.nm new ommm v.00 H.mo mmm. mum. m.ao m>HHQIwHQSOQ .Uomm maom maom .cmumlcoc .cmnmlcoc .cmum .G.c mumu mufl on mLz ou oncom ou MN coma .t.a IHmHom pcmoumm unwoumm uswoumm Inmaom .>Hmmno usmoumm .maoflum>ummno Hmoflmoaouho mo mnmfiesm .m manna 30 not located at the pole indicated by the spindle axis, but was positioned to one side of that pole. Usually, this difference between spindle axis and granular axis was slight. However, once the granular mass approached the equatorial region, it had to be considered to be unpolarized. Thus, these nonpolarized primary spermatocytes may have actually exhibited polarization prior to disturbance of the rigid Spindle and granular position by squashing. The observed nondisjunction rate was higher when sc4sc8 was in the presence of §Q at both 280 and 18°C. No nondisjunctional events were observed in the gagg class. At 18°C, the rates of nondisjunction in both sc4sc8 and ”Double-Drive" primary Spermatocytes was lower than those observed at 28°C. In Basc-y Y cells, the sex chromosomes tended to undergo anaphase movement at the same time as the autosomes. In sc4sc8 and ”Double-Drive”, however, the movement of the X and Y to the poles was generally precocious with respect to that of the autosomes. This undoubtedly is due to lower frequency of synaptic association because of the reduced X-Y homology with the sc4sc8 gross heterochramatic deficiency. The anaphase behavior with respect to the position of the granules in disjunctional or nondisjunctional polarized cells did not appear to be random. In the 28°C "Double- Drive" disjunctional class, sc4sc8 travelled to the non- granular pole with a frequency of 61%. At 18°C, this fre- quency was reduced to 58%. At 28°C, the sc4sc8 chromosome 31 oriented to the non-granular pole with a frequency of 64% of the time. At 18°C, this frequency was reduced to 55%. The nullo pole of nondisjunctional cells was usually that pole without the granu1ar mass. In 28°C "Double—Drive" nondisjunctional cells, the pole to which sc4sc8 X and Y travelled was the granular pole in 65% of such events. At 18°C, this frequency was 61%. The corresponding frequencies for sc4sc8 were 64% at 28°C, and 64% at 18°C. The behavior of the Basc X appeared to be random with reSpect to its orientation to the granular pole. Genetic Tests of sc4sc8 and ”Double-Drive" Tables 9, 10 and 11 summarize the results of the progeny tests of the sibs of males sacrificed for cytological study. The 3_values exhibited by "Double-Drive" males increased from 91.3% at 28°C to 98.9%Iat 18°C. The corresponding over-all rates of nondisjunction decreased from .512 at 28°C to .305 at 18°C. When the sc4sc8 drive system operated in the absence of §Q the over-all rates of nondisjunction were lower than with §Q, At 28°C and 18°C, the nondisjunction frequencies were .323 and .149 respectively. These results are in accord with those of Zimmering (1963), with the frequency of non- disjunction being reduced by almost half at 18°C (Zimmering cultured his sc4sc8 males at 180 and 26°C.) The presence of §Q_ostensib1y increases the efficiency of the sc4sc8 meiotic drive system at both high and low 2 3 «mag mom. hog. mem. m.mm om m NHH -Hvaa m 5H Nana Hoaa oma nmmv Nam. mmq. mam. m.Hm 00H q ma mmom Ho mod mmva Hmw 0mm .@.: Ham .©.: .0.: m m w m x \0 MN > \D Hmuoa IH0>O 33:0 Om x m + \o m + \b m + m .mEme 33:0 33:0 Gm Gm 39:0 39:0 mm +>N Gm :.0>HHQI0HQSOQ: I mumwu >:0moum mo ammEEDm .m magma 33 Table 10. "Double-Drive" segregation frequencies. Segregation Frequency I II 28°C. 18°C. sc4sc8 §Q_ .427 .336 Y cnbw .004 .034 4 8 sc sc cnbw .001 .014 F‘ ' Y _S_p_ .263 .104 “ nullo §Q_ .272 .469 sc4sc8-Y cnbw .005 .038 nullo cnbw .001 .001 sc4sc8-Y .89 .028 .004 b3 Table 11. Summary of progeny tests sc4sc8,y. L -_ cnbw cnbw cnbw cnbw rate of Temp. x/x+y ‘o/o+xy Total Yy+cf y 2 y cf y+ i n.d.* 28° 490 1722 1020 36 .323 .778 .966 3268 18° 399 487 99 54 .149 .550 .647 1039 34 temperatures. Yet, in the "Double-Drive" situation, the meiotic drive of S9 increases at 180 whereas that of sc4sc8 decreases. Without sc4sc8, this §Q(+/y+Y;SD;72 + +) has a SD cnbw 5 value of 99.6 at 28°C and 99.1 at 18°C. Therefore, the presence of the sc4sc8 system at 28°C appears to effectively reduce the drive of SD while increasing the drive of sc4sc8. '5. At 18°C, the interaction of the systems increases sc4sc8 drive while the g_of SD is apparently unaffected. The relative frequencies of the reciprocal products of disjunctional and nondisjunctional events at 28°C were: 3‘ I} X/X+Y O/O+XY "Double-Drive" .718 .917 sc4sc8 .778 .966 Thus, the drive of the disjunctional sc4sc8 actually de— creased in the presence of S2, as did the "drive” of the non- disjunctional nullo product. Therefore, at 28°C, the drive of both sc4sc8 and §Q_are lowered. At 18°C, the results differ from those at 28°C. X/X+Y O/O+XY "Double-Drive“ .616 .893 sc4sc8 .550 .647 The drives of both disjunctional and nondisjunctional products I 4 8 I I are increased when the sc sc system is in the presence of §Q_at 18°C. Therefore, at 18°C, the drive of SD is apparently unaffected by the presence of sc4sc8, while that of sc4sc8 is heightened. 35 Table 10 shows the frequency of segregation of dis— junctional and nondisjunctional sc4sc8 chromosomes with S2 or cnbw, Considering disjunctional products, the association of sc4sc8 and SD decreases from .427 at 28°C to .336 at 18°C, while the reciprocal association of y+Y with SD+ cnbw in- creased from .004 to .034. Accordingly, the segregation of sc4sc8 with SD+ cnbw increased from .001 at 28°C to .014 at 18°C. From genetic evidence, it appears that the sc4sc8 X 7 and S2 arrive at the functional pole (F pole) together with greater frequency at 28°C than at 180C. [‘97. .; $.‘il’5’f‘.’M-- ' { . In the case of nondisjunctional products, the fre: Q l quency with which the §Q_segregates to the nullo pole increases at 18°C (.272 at 28°C vs. .469 at 18°c), as does the fre- quency of sc4sc8, Y - Egby_association. The segregation frequency of 9gbw_to the nullo pole remains the same (.001) at 18°C and 28°C. That of sc4sc8, Y and §Q decreases from .028 to .004. The segregation of the nondisjunctional product and §Q at high and low temperatures present a different picture than SQ segregation with disjunctional products. Both nullo- §2_and ggfgg?X,Y3gn§w segregation frequencies increase at 18°C. Apparently, the probability of §Q_reaching the nullo F pole and of gnbw arriving with XY at the F pole increases at 18°C. The repulsion between SD and the sc4sc8X1y+Y chromosomes increases as the temperature is lowered. The postulated homology between the SQ complex and X (Hiraizumi, 1967) may be supported by these data. The abbreviated 36 sc4sc8 X has less homology with the Y than a normal X (Cooper, 1959). However, the sc4sc8 X and y+Y together may bear enough homology to the §Q_complex to cause such repulsion. Correlation of Cytological Studies and Genetic Evidence Tables 12 and 13 compare cytological observations with resultant progeny data. The frequency of nondisjunction f1 in all primary spermatocytes and that exhibited in the F2 are in agreement. A 3 x 2 X2 test (Table 14) of rate of disjunction versus the presence and location of the granules, 1.2-..- ‘ gives a X2 value of 2.72. Therefore, the probability is about 30% that this distribution would occur in populations with homogeneous means. These data support Yanders gt a; (1968) in their conclusion that meiotically driven chromosomes and the bacterial cellular inclusions are responding to the same cellular polarity, and that this polarity is associated with the determination of functional and non-functional gametes at Meiosis I (Peacock and Erickson, 1965). These bacterial inclusions appear to serve as an index of the normal polarity of a spermatocyte. Yanders gt 31, derived the following parameter, p, as an estimate of the probability that the bacteria will be located at the nonfunctional pole of the anaphase I Spindle: s=pt+(l-p)(l- t) Where §.is the observed frequency of occurrence of the bacteria with the Y chromosome in disjunctional anaphases, 37 n at : m>HHQImHQ50Q: Inn 99.1% :OHu0:shmH©:o: u .p.:* hwo. mwo. 0mm. mmm. oma. hva. mmm. mug mmm. moo. 0H0. Hmm. mom. mom. wmm. than oma mom. H¢w. mph. New. wmv. mmm. mmm. mug ham. amp. man. how. mmm. mam. mmm. than 0mm mmumfimm mmumemm Hm 0H0: “was maamo 8.0.: 00H::mum I:0H00::nmflp I:mum :0: @0NHHmaom 8.0.: «.U.: H80 :H Oaas: ou Mx :H m0m¢0m ou m0m¢0m :H 8.0.: 0Hu0:0w IHmoHoumu .m:OHum>H0on 0H00:0m ©:m Hm0flmoaou>0 mo :OHumawnnou .NH waflmfi 38 90>AHQI0HQDOQ: u .Q.Q*¥ 3830588682 u 6.2.. 0mm. mo.a hdo. 0mm. mew. mmm. mug oma qmo. mvm. mam. mam. moo. Hmm. 8*.Q.n oma Hmo. mmh. mom. mun. ave. Nvo. mlq 0mm Hmm. men. ham. man. Hmm. boo. 88.9.Q owm a Q 0H0: 0H0: amas:mnml:o: on Hma::muml:o: on Hm:0Hu0::n Hm:0Hu Aug Auv Amy Amy Imetcoz Iucsnmflo xx + 0\o w + x\x can + 0\o w + x\x *.Q.z Iommwmmm *.Q.z Hm:0600:snmfin m:0aum>ummno 0Huw:00 m:oaum>nwmno HMUHmoHou>U .muuscoum Hm:0Hu0:shmflp:o: p:m Hm:0H00:Sanp How m.H000&mHmm 0:0 How mwsam> .ma mamas 39 Table 14. Test for homogeneity of means. 2 II . u 0 3 x 2 X Test, Double-Drive at 28 C. 2 (T/nz) —-§i X2 = Zn § (1 - §) Polar. Non—polar Agranular Total cells cells cells F3 Disjunc. cells 84 43 19 146 (G) Nondisj. cells 106 33 25 164 . Sample PJ size 190 76 44 310 (Zn) Item Computation = 2 means = Mean .792 .566 .432 .47096 y 1.790 T2 7056 1849 361 21316 G2 ZTZ/n = 69.45 TZ/n 37.14 24.33 7.98 68.76 02/2n § ll-§) .253442 2(T2/n - GZ/Zn) .69 2 X 2.72 40 or with the X and Y chromosomes in nondisjunctional anaphases (see Table 13), §.is the coefficient of meiotic drive for X or nullo gametes from disjunctional or nondisjunctional gamete classes respectively. The calculated p_va1ues are given in Table 13. The mean value of p for disjunctional and non- 8 disjunctional products of sc4sc males at 28°C is .703. Yanders §t_§1 (1968) reported a similar é value (.741) for sc4sc8 at 25-26OC. At 28°C, the p_va1ues for disjunctional and nondisjunctional events in ”Double-Drive” and sc4sc8 are comparable. At 18°C, sc4sc8 gave 2 values in close agreement. In "Double-Drive" at 28°C, however, the pro- bability that the polarized bacteria are located at the nonfunctional pole is .849 in disjunctional gametes, and .634 in nondisjunctional gametes. In all cases, the probability of the bacteria being at the nonfunctional pole is greater in disjunctional gametes than in nondisjunctional gametes. The p_va1ue for sc4sc8 at 18°C are in close agree- ment (difference = .017), as are the p values for "Double- Drive" at 28°C (difference = .064). The differences between nondisjunctional and disjunctional p values for "Double— Drive” at 18°C and sc4sc8 at 28°C are .215 and .104 respec- tively. The probability that the nonfunctional pole will exhibit granules appears to be influenced by environmental conditions such as temperature, and the extent and direction of this influence is dependent upon the chromosomes in the 41 drive system (or systems). The extent of the influence of temperature may differ in disjunctional and nondisjunctional gametes. .lJ l-t:-n'k ’ -Am'l‘ 5.“.- VI. DISCUSSION The Effect of Irradiation on SD The depression of the k_va1ues in the sperm batches of irradiated males indicates that the mechanism of §Q is disturbed by irradiation. The period of sensitivity occurs prior to Anaphase I, probably during the period of synapsis at zygotene of Prophase I. The percentage of aberrations observed per sperm batch is inversely correlated with the k values exhibited. Since the probable origin of these aberrations is irradiation—induced chromatid breakage and subsequent rejoining of non—sister chromatids, the chroma- tids involved would most likely be in synaptic association. The sperm batches containing the greatest proportion of chroma- tids affected by these events also exhibited the lowest 3 values, suggesting that the SQ mechanism was most sensitive to irradiation at the time of synaptic pairing. This interpretation is supported by the work of Sandler, Hiraizumi, and Sandler (1959) who found that inhibition of pairing at the §2_Ag(§9) region by inversions in the §Q+ homologue greatly depressed the drive of S2, Irradiation at synapsis may reduce the effectiveness of SQ by disturbing the intimate association of the chromatids. Mange (1968) found the k_values of §2_ma1es most reduced 7 to 11 days following treatment of temperature- 42 43 sensitive SQ males for a 24 hour period at 300 or 19°C. The irradiation—induced reduction of k_va1ues in these experiments was most pronounced 7 to 10 days after treat- ment. Therefore, the period during which the §Q_mechanism is sensitive to temperature appears to coincide with the irradiation-sensitive stage. The k_value depression observed after irradiation of §Q;12 is comparable to that reported by Mange after cold shock of §2112 for 24 hours at 19°C. (82:12 was reported to be insensitive to heat treatment.) Storage of both treated and control males affected the k_of SQ. Only the first sperm batch of treated males carried aberration products. The sperm in later batches were most probably not yet primary Spermatocytes at the time of irradiation. The erratic k values of both the control and experimental groups can be attributed to prolonged storage of the adult male without mating. Storage apparently "unstabilizes" §Q. Why storage should reduce the stability of §Q_is not clear, but sexual abstinence could retard the rate of spermiogenesis, and prolongation of the primary Spermatocyte stage could cause disorientation at Anaphase I. The sex ratios (QR/total) of gg_by_progeny of SDZQn bw males were in excess of 60% in all groups. Hiraizumi (1967) reported such an increase in the proportion of females in £2.22! with a reciprocal decrease in the proportion of females in SD progeny. Because of this, he postulated a homology between the X chromosome and the SQ §g(§2) §tfl§2) complex, and this type of homology would account for the current ‘5 1" (‘2‘? 44 results. This would imply that a degree of repulsion exists between the SQ bearing II and the X resulting in their ten- dency to segregate at Anaphase I to opposite poles. However, a reciprocal decrease in the proportion of SD females was not observed in these data. The S2 progeny and the homozygous g§_bw_stock exhibit similar sex ratios of about 50%. Since Sthas been maintained by back crossing '5 it to En by every generation, the §Q_bearing chromosome II should be essentially the only difference between the two stocks. The absence of a reciprocal depression of the sex J ratio in the two classes is difficult to reconcile with Hiraizumi's original homology hypothesis, which implied an "effective pairing” before Anaphase I. To account for these data, a modification concerning the chromosomal sequence of Anaphase I segregation is proposed: 1) prgg precedes the sex chromosomes to the functional or nonfunctional pole at Anaphase I, the probabilities of the X or Y reaching that pole are equal. That is, the arrival of the X at that pole is effectively a random and independent event. 2) If the sex chromosomes undergo Anaphase I segregation prior to the autosomes. and the X chromosome travels to the F-pole, the probability of §Q orienting to the F-pole is reduced. The reciprocal Y; §Q gamete is nonfunctional. Effectively, this is unilateral repulsion. The chromosome does not ”recognize" sufficient homology with the §Qpchromosome to disorient its movement at Anaphase I. The 45 SD complex on chromosome II, however, is somewhat repelled by an X-chromosome-bearing pole. A reciprocal decrease in the §Q_sex ratio would therefore not occur. The S f gn,bw_ chromosome would have an increased potential of reaching the F-pole if the X preceded it. The reciprocal Y7 §2_gamete is lost. If the X segregated precociously to the nonfunction- al pole, the strong drive of the SQ chromosome to orient to- wards the F-pole would be unaltered, or slightly increased. If the drive of §Q is somewhat increased by repulsion from an X-bearing nonfunctional pole, the subsequent increase in the proportion of functional Y: §2 gametes would compensate for the loss of Y; §Q_nonfunctiona1 gametes resulting from the repulsion of §Q_toward an X-bearing functional pole. Relationships of Cytological and Genetic Data Comparison of the rates of nondisjunction observed in primary spermatocytes and recovered in progeny of sib males indicates that the cytological and genetic data are measurements of the same meiotic event. Both methods indi- cated 1) an increase in the frequency of nondisjunction in sc4sc8 when in the presence of §Q_in "Double-Drive", at 180 and 28°C, 2) a decrease in nondisjunctional events in both sc4sc8 and "Double-Drive" at 18°C and 3) comparable rates of nondisjunction in both sc4sc8 and "Double-Drive" at 180 and 28°C. Yanders et a1 (1968) concluded that the polarization of the bacteria at Meiosis I was an independent response to 46 a cytoplasmic gradient which regularly exists in the primary spermatocyte. This gradient renders the meiotic poles un- equal, supporting the cytological model for meiotic drive proposed by Peacodk and Erickson (1965). Their function- ality hypothesis requires a predetermined polarity of the primary spermatocyte, with only one pole yielding functional sperm. 0n the basis of the p values calculated (p,= the probability that the bacteria will be located at the nonfunctional pole at Anaphase I, Yanders et_§1, 1968), the observed frequency of association of one or the other recipro- cal class of disjunctional or nondisjunctional events in sc4sc8 is a reasonable estimate of the genetic recovery of that class. The probability that the pole exhibiting polarized bacteria will be nonfunctional is slightly lower in non- disjunctional cells. In the cases of sc4sc8 at 18°C and "Double-Drive" at 28°C, the p_va1ues for disjunctional and nondisjunctional cells differ by only .017 and .064 re- spectively. In sc4sc8 at 28°C and in "Double—Drive" at 18°C, the differences are somewhat greater, .104 and .215. The p_value differences do not correlate with the characteristic differential in recovery of nondisjunctional reciprocal products reported by Zimmering (1963) and Peacock (1965), and observed in both sc4sc8 and "Double-Drive" at both temperatures. Although the propensity of the bacteria to be located at the nonfunctional pole at Anaphase I is significant in every category, there appears to be a variance with 47 temperature, chromosomal constitution, and segregation be- havior at Anaphase I. The 3 x 2 X2 test for homogeneity of means indicated that polar, non-polar, and agranular cells did not differ significantly in frequencies of nondisjunction. Therefore, the polarized cells scored for the association of bacteria with the reciprocal products of disjunctional and non- disjunctional events can be considered to be representative of the population in general. In addition to the cytological evidence that a polarity exists in the primary spermatocyte prior to Anaphase I movement, the genetic tests of sc4sc8 and "Double-Drive" support the hypothesis that functionality is determined prior to chromosomal segregation. If the functional pole were determined by the driving chromosome itself, sc4sc8 and §2_wou1d be in a competitive situation. Yet, at 18°C. the SD 3 value in ”Double-Drive" is characteristic of the drive of this SQ alone (.989), and the drive of sc4sc8 is increased in the presence of S2, At 28°C, the k_of §Q_ in "Double-Drive“ was decreased (.917), as was the drive of sc4sc8. These results indicate that the §Q_and sc4sc8 systems were both responding to a gradient in the spermato- cyte associated with functionality. Hartl, Hiraizumi, and Crow (1967) proposed that the mechanism of §Q_was the production of dysfunctional ng sperms. Such a mechanism would be independent of any polarity gradient in the primary Spermatocyte. This model is formally 48 equivalent to the chromosome breakage hypothesis concerning g2 (Crow 33 31, 1962) but does not require cytological evidence of actual breakage events. If SQ action is independent of the cytoplasmic gradient to which sc4sc8 responds, the drive of sc4sc8 should be unaffected by that of S2, The occurrence of sc4sc8 with §Q_or §Q+ would be randomly determined, those sperm bearing sc4sc87§2f being rendered dysfunctional. The present data indicate that the drives of these two systems are indeed related, with sc4sc8 exhibiting an in-' creased drive when the 5 of £1; is higher (at 18°C) and a diminished drive when §Qfs k.is lower (at 28°C). This is not compatible with the dysfunctionality hypothesis. Sandler, Lindsley, Nicoletti, and Trippa (1968) consider S2 to be in a class of meiotic mutants which dis- turb normal meiotic behavior of chromosomes. In their germinal cycle model, §Qfs action would most likely be exerted after centromeric association and the onset of pair- ing. The S2 locus must pair with the ng locus on its homologue in order to effect segregation-distortion (Sandler, Hiraizumi, and Sandler, 1959) and subsequent chromosomal disjunction is normal (Hiraizumi and Hartl, 1968). The aberrant segregation of the sc4sc8 chromosome is probably due to its gross heterochromatic deletions. Deleted X chromosomes characteristically exhibit greatly reduced pairing with the Y and nondisjunction (Lindsley and Sandler, 1958). It is possible, however, that a locus 49 (or loci) for normal genetic control of sex chromosome dis- junction and Anaphase I segregation is included in the segment deleted from sc4sc8. While both S2 and sc4sc8 exhibit aberrant segrega- tion, they do so for different reasons. In the case of sc4sc8, the deletion of proximal heterochromatin produces a chromosome which is much smaller than the normal X, and con- siderably smaller than the Y. It has been shown that when the homologues differ in size, the smaller of the two is recovered more frequently (Novitski, 1956a). On the other hand, there is no difference in size between SD and its-SQ+ homologue, and the SQ locus itself, presumably a single site in the heterochromatin, is responsible for the aberrant segregation. This is a qualitative difference in homologues, while that of sc4sc8 is quantitative. Nevertheless, both §Q_and sc4sc8 exhibit a pronounced meiotic drive, even though the mechanisms initiating the drives are dissimilar. The model which best fits these results is the functional-nonfunctional pole hypothesis rather than the dysfunction hypothesis. If the chromosomes themselves determined polar functionality, as is required by the dys- function model, the two driving systems would compete and one or both drives would be diminished. The present data do not indicate such competition. The cytologically ob- served drive of sc4sc8 away from the granular pole at Anaphase I and its comparable genetic recovery support the existence of a predetermined functional-nonfunctional polarity 50 in the primary spermatocyte. Although the §23bearing chromosome II is not cytologically distinguishable from its §Qf homologue, the concomitant elevation and depression of the §Q_k value and sc4sc8 evident at 18°C and 28°C indicates that the mechanism of SD is not independent of the polarity gradient to which sc4sc8 responds. V. SUMMARY The research was designed l) to ascertain the effect of irradiation at the onset of meiosis on Segregation- distorter in order to determine the meiotic stage during which the mechanism of §Q_is sensitive, and 2) to observe the meiotic drive systems of SD and the sc4sc8 X together (“Double-Drive") both cytologically and genetically at 180 and 28°C. Analysis of the interaction of these systems and correlation of the nonrandom polar segregation of sc4sc8 with its genetic recovery tested the hypothesis that the mechanism of both drive systems involves a predetermined functional-nonfunctional polarity which is independent of the drive systems which respond to it. In the first study, irradiation treatments were administered to very late third instar male §Q_1arvae. To observe the effects of sperm storage, part of the experi- mental and control groups were aged after eclosion as virgins. The first four daily broods from unstored irradiated males exhibited depressed k values. The percentage of aberrations and the depression of the k values per brood appear to be positively correlated. Using the proportion of aberration products as an index, stored males appear to transfer few sperm which were in meiotic or premeiotic stages at the time of treatment. 51 52 Depressed k_values were exhibited by both irradiated and control groups which were stored. Lack of mating activity and possibly resultant prolongation of Meiosis I appear to "unstabilize” and depress the drive of SD, The sex ratios (gg/total) of gg_bw_progeny in experi— mental and control groups were in excess of 60%: A modifi— cation of Hiraizumi's §Q¢X chromosome homology hypothesis is presented to account for the lack of a reciprocal de- crease in the sex ratios of §Q_progeny. In the study of "Double—Drive", the nonrandom segre- gation of sc4sc8 or the nondisjunctional nullo product away from the bacterial pole correlated with genetic recovery, supporting the hypothesis that a predetermined functional- nonfunctional polarity exists in the primary spermatocyte. At 18°C, sc4sc8 expresses increased drive when in the pre- sence of S2. Both drives are slightly reduced when together at 28°C. Thus, the data indicate that the drive systems of §Q_and sc4sc8 are not competitive in their interaction, and that the mechanism of SD is not independent of that to which sc4sc8 responds. BIBLIOGRAPHY II... _ _r 5161.11.31 P BIBLIOGRAPHY Beadle, G. W., and B. Ephrussi, 1936 ”The Differentiation of Eye Pigments in Drosophila as Studied by Transplantation." Genetics 21: 225-247. Carpenter, J. M., 1950 "A new semi-synthetic food medium for Drosophila." Drosophila Information Serv. 24:96. Cooper, K., 1950 "Normal spermatogenesis in Drosophila", In: Biology of Drosophila, M. 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Hiraizumi, 1959 "Meiotic drive in natural populations of Drosophila melanogaster. II. Genetic variation at the Segregation-distortion locus." P.NuA.S. U.S. 45: 1412-28 1960a ”IV. Instability at the §Q_locus." Genetics 45: 1269-1287. 1961b. "VIII. Conditional Segregation-distortion: A possible nonallelic heritable aging effect on the phenomenon of Segregation-distortion." Canadian Journal of Genetics and Cytology 3:34-36. Sandler, L., Y. Hiraizumi, and I. Sandler, 1959 ”Meiotic drive in natural populations of Drosophila melanogaster. I. The cytogenetic basis of segregation-distortion.” Genetics 44: 233-50. Sandler, L., D. L. Lindsley, B. Nicoletti, and G. Trippa, 1968 "Mutants affecting meiosis in natural populations of Drosophila melanogaster." Genetics 60: 525-558. Yanders, A. F., J. G. Brewen, W. J. Peacodk, and D. J. Goodchild, 1968 "Meiotic drive and visible polarity in Drosophila spermatocytes.” Genetics 59: 245—253. Zimmering, S., 1963 ”The effect of temperature on meiotic loss of Y chromosomes in the male Drosophila.” Genetics 48: 133-138. Zimmering, S., and R. E. Green, 1965 "Temperature dependent transmission rate of a univalent X chromosome intzhe male Drosophila melanogaster." Canadian Journal of Genetics and Cytology 7: 453-456. Zimmering, S., and M. Perlman, 1962 "Mbdification of abnormal gametic ratios in Drosophila. III. Probable time of the A-type effect in Bar of Stone translocation males.” Canadian Journal of Genetics and Cytology 4: 333-336. A PPEN’D IX APPENDIX Basc constitution: In(1)SCSlLSC8R+S, scSlscewa B. synthesis: Muller synonym: M-S: Muller-5 references: Spencer and Stern, 1948, Genetics 33: 43-74 properties: Male and homozygous female viable and fertile; X/O male poorly viable, variegated for y, ac, and probably 1(1)Jl. Suppresses crossing over in X, but less so than Binsc because In(1)S = In(1)6A1-3;1OFlO-11A1 less effective than In(l)d1-49 =In(l)4D7-E1;llF2-4. Routinely used in detection of sex-linked recessive lethals. cinnabar location: 2-57.5 origin: Spontaneous discoverer: Clausen, 20i8. references: 1924, J. Exptl. Zool. 38: 423-36 phenotype: Eye color bright red, like v or st. Ocelli colorless. Eye color darkens with age, but ocelli remain colorless. Larval Malpighian tubes pale yellow (Beadle, 1937, Genetics 22: 587-611). Nonautonomous in development of pigment of transplanted eye disks (Beadle and Ephrussi, 1936, Genetics 21: 230), cn blocks conversion of kynurenine to 3-hydroxyhy2urenine, which has been identified as the on hormone (Butenandt, Weidel, and Schlossberger, 1949, Z. Naturforsch. 4b: 242-44). RKl. cytology: Proximal to 44C, based on its inclusion in Dp(2;3)P32 = Dp(2:3)41A;42D-E;44C-D;89D7-E1 (E. B. Lewis). brown location: 2-104.5 discoverer: Waaler, 19j15. references: 1921, Hereditas 2: 391-94. Sturtevant and Beadle, 1939, An Introduction to Genetics, Saunders, p. 64 (fig.). phenotype: Eye color light brownish wine on emergence, darkening to garnet. Red pigments lacking; ommo- chromes at 87 percent normal level (No1te, 1954, 58 59 J. Genet. 52: 111-26). Adult testes and vasa colorless. Larval Malpighian tubules pale yellow (Beadle, 1937, Genetics 22: 587-611). Produces white eyes in combination with v, cn, or st. Eye color autonomous when transplanted into wild-type host (Beadle and Ephrussi, 1936, Genetics 21: 230). RKl. cytology: Placed between 59D4 and 59E1 by Bridges [1937, Cytologia (Tokyo), Fujii JUb. Vol. 2: 745- 55], on the basis of its exclusion from the inner inversion of In(2LR)wal = In(2LR)21C8— D1;60Dl-2 + In(2LR)4OF:59D4—El and its inclusion in In(2R)wa De2 = In(2R)4lA-B759D6-El. Based on the study of bw rearrangements, Slatis (1955, Genetics 40: 5-23) tentatively places bw in 59D9, 10, or 11. other information: Separable into at least two sub- units by recombination with bw and bw75 about 0.001 units to the left of bw59 and hw81 (Divelbiss, 1961, Genetics 46: 861). 2: yellow location: 1-0.0. origin: Spontaneous. discoverer: E. M. Wallace, 11a references: Morgan and Bridges, 1916, Carnegie Inst. wash. Publ. No. 237: 27. phenotype: Body color yellow; hairs and bristles brown with yellow tips. Wing veins and hairs yellow. Tyrosinase formed in adults (Horowitz). For the most part, y is autonomous in mosaics; tissue, however, over limited distances there is some nonautonomy [Hannah, 1953, J. Exptl. Zool. 123: 523-60 (fig.)]. Larval setae and mouth parts yellow to brown, hence distinguishable from the dark brown of wild type (Brehme, 1937, Proc. Soc. Exptl. Biol. Med. 37: 578-80; 1941, Proc. Natl. Acad. Sci. U.S. 27: 254-61). RKl cytology: Placed in region 1A5-8 on basis of its being carried by the XDBP element of T(l;3)sc260-20 = T(1:3)lA8-Bl;61A1-2 and by Dp(l;f)sc260-27 = Df(1;f)1A8-Bl;19F, but not being lost from Df(l)260-5 = Df(1)lA4-5 (Sutton, 1943, Genetics 28: 210-17). 4L 8R . . In(l)sc sc : InverSlon(1) scute-4 Left scute-8 nght cytology: In(l)1B3-4;19F—20C1L1B2-3:20B-D1R: duplicated for 1B3, mitotic chromosomes deficient for the proximal third of hD, all of hC and hB, and the distal majority of hA (Cooper, 1959, Chromosoma 10: 525-88). About 0.6 the length of a normal X at metaphase. 6O origin: Recombinant containing left end of In(1)sc4 and right end of In(1)sc8. discoverer: Gershenson. references: 1933, J. Genet. 28: 297-313 1933, Biol. Zh. (Moscow) 2: 145-59, 419-24. genetics: Duplicated for the sc locus, carrying both so and sc ; deficient for the bb locus and the nucleolus organizer [i.e., Df(l)bbG]. Shown by Ritossa and Spiegelmann (1965, Proc. Natl. Acad. Sci. U.S. 53: 737-45) to be deficient for all the DNA that is complementary to ribosomal RNA present in a ha loid chromosome set. In the male, In(1)sc LSCBR frequently fails to pair with the Y and when it does the unpaired X and Y usually proceed to the same pole (Peacock, 1965, Genetics 51: 573-83). Furthermore, reciprocal meiotic products are not recovered with equal frequency, which Peacock interpreted as the result of non-random orientation of the first meiotic division with respect to the postulated functional pole of the primary spermatocyte. Irregularities in meiotic behavior of In(1)sc4Lsc8R in the male are affected by the Y chromosome present (Peacock, 1965) and the temperature at which meiosis occurs 3Zim§ering, 1963, Genetics 48: 133-38). In(1)sc LSc R/Y/Y male gives quite regular segre- gation of the two Y's and low recovery of the X. "Illlllllllll‘llll