Experiments on chromosome elimination in the gall midge, Mayetiola

/ . Embryo!, exp. Morph. Vol. 24, 2, pp. 257-286, 1970 2 5 7Printed in Great Britain

Experiments on chromosome elimination in thegall midge, Mayetiola destructor

ByC. R. BANTOCK1

From the Department of Zoology, Oxford University

SUMMARYCleavage in Cecidomyidae (Diptera) is characterized by the elimination of chromosomes

from presumptive somatic nuclei. The full chromosome complement is kept by the germ-linenuclei.

The course of cleavage in Mayetiola destructor (Say) is described. After the fourth divisiontwo nuclei lie in the posterior polar-plasm and become associated with polar granules, andfourteen nuclei lie in the rest of the cytoplasm. All the nuclei possess about forty chromosomes.During the fifth division the posterior nuclei do not divide and the polar-plasm becomesconstricted to form primordial germ cells (pole cells). The remaining fourteen nuclei divideand lose about thirty-two chromosomes so that twenty-eight nuclei are formed containingonly eight chromosomes. These are the presumptive somatic nuclei. During subsequentdivisions the pole cell nuclei retain the full chromosome number; these divisions occur lessfrequently than those of the somatic nuclei.

Experiments were performed on early embryonic stages to elucidate the properties of theposterior end during the time that chromosome elimination was taking place from the pre-sumptive somatic nuclei. Ultraviolet irradiation, constriction, and centrifugation techniqueswere used.

The polar granules are concerned with the non-division of the germ-cell nuclei during thefifth division, since if the granules are dispersed by centrifugation, or if nuclei are preventedby constriction from coming into contact with them before the fifth division, all the nucleidivide with chromosome elimination at this division. With each technique it is possible toobtain embryos possessing germ cells with only eight chromosomes in their nuclei.

Individuals possessing germ-line nuclei with only eight chromosomes were allowed todevelop to maturity. Abnormalities were confined to the germ cells only and were the sameregardless of which technique had been used to produce the deficient germ line. An ovarycontaining germ-cell nuclei with only eight chromosomes is unable to form both oocytesand nurse cells. A testis containing germ-cell nuclei with only eight chromosomes is unableto form spermatocytes but cells which come to resemble gametes are formed. Experimentalmales and females are both sterile.

The results are discussed in relation to other experimental work on Cecidomyidae and thefollowing main conclusions are reached: (a) the polar granules are responsible for preventingan irreversible loss of chromosomes from the germ-cell nuclei by preventing the mitosis ofthese nuclei during the fifth division; (b) the chromosomes normally retained in the germ lineare required for gametogenesis, particularly for oogenesis.

The significance of chromosome elimination is discussed.1 Author's address: Department of Biology and Geology, Northern Polytechnic, Holloway

Road, London, N.7., U.K.

258 C. R. BANTOCK

INTRODUCTION

In some animals the germ cells originate during embryonic cleavage and it iswell established that experimental procedures which interfere with the normalassociation between the germ-line nuclei and the cytoplasm these occupy canresult in sterility (e.g. Geigy, 1931).

In the Cecidomyidae (Diptera) germ cells form as a result of one or more ofthe posterior cleavage nuclei migrating into the cytoplasm at the posterior endof the embryo (polar-plasm) and the polar-plasm becoming constricted off toform germ cells (pole cells). There is no evidence that in this family of insectsthe pole cells give rise to any tissue other than the germ cells.

Synchronous with, or immediately after, the formation of pole cells in Ceci-domyidae are atypical mitoses of all of the presumptive somatic nuclei. Theseenter mitosis and a large but specific number of the chromosomes remains atthe equator at anaphase and fails to enter the daughter nuclei. This is known aschromosome elimination and was first observed in Miastor by Kahle (1908). Thechromosomes which are eliminated are designated E chromosomes, and theremainder S chromosomes. Somatic nuclei have only the S number and theprimordial germ cells have the full E+S number (White, 1950).

In many male cecidomyid embryos there is a further elimination of chromo-somes from the presumptive somatic nuclei. White (1950) considers that thisdifference between the elimination pattern of the two sexes is important in sexdetermination in Cecidomyidae.

The formation of pole cells in Cecidomyidae is marked not only by theelimination of chromosomes from the presumptive somatic nuclei but also bythe onset of a lower frequency of mitosis of the germ-line nuclei. Chromosomeelimination is a particularly clear example of nuclear differentiation at thegenetic level and has been subjected to experimental analysis by Geyer-Duszynska (1959, 1961, 1966), by Nicklas (1959) and by the present author(1961). The aim of the present work is an attempt to throw some light on thecauses of the retention of the E chromosomes by the germ-line cells, on thefactors causing the germ cells to divide less frequently than the somatic nuclei,and on the functions of the E chromosomes. The Hessian Fly Mayetiola de-structor (Say) was chosen since it was found to be possible to breed this speciescontinuously through the year.

MATERIAL AND METHODS

The eggs of Mayetiola are laid in longitudinal rows on the leaves of wheatplants in the 2-leaf stage. The eggs are deposited always with the posterior endof each nearest the axil of the leaf. Generally the eggs from any one female willdevelop into adults of only one sex. The eggs hatch in 3-5 days and the larvaemigrate to a position under the ligule. This position is maintained throughoutlarval and pupal life and the last larval and pupal instars are spent in a

Chromosome elimination experiments 259

puparium. The life-cycle is completed in 47-50 days during the summer monthsin England. Under natural conditions there are three generations a year, thelast generation overwintering in the puparia. Emergence takes place early inthe morning; egg-laying starts within an hour or two of mating and may con-tinue for as long as 48 h.

Breeding techniques

Development can be temporarily arrested by storing puparia at 5 °C in Petridishes containing moist filter paper, but it was not possible to prevent emergenceonce pupation had occurred. When adults were required puparia were trans-ferred to glass cylinders 10-2 cm in diameter closed at the top end by muslin-covered metal rings, the cylinders resting on moistened peat fibre and kept at20 °C. Adults emerged in 3—14 days. Those adults required for maintaining thestocks were transferred to potted Peko wheat plants in the 2-leaf stage. Theplants were enclosed by glass cylinders closed with muslin at the top. There isa mortality rate of approximately 40% in young larvae before they becomeestablished under the ligule; they are particularly susceptible to desiccation.Humidity was maintained in the cylinders by covering the tops with wet clothswhen oviposition was completed. The cylinders were removed and the plantscovered with muslin bags when all the eggs had disappeared from the leaves.

When eggs were required for experimental purposes short pieces of leafwere placed in glass tubes containing mated females. Embryos and younglarvae were handled with silver-plated entomological pins set in matchsticks.Post-experimental embryos required for analysis in later stages in the life-cyclewere transferred to a drop of water in the axil of a leaf, from which the larvaewere able to take up their normal position. All experimental embryos were kepta t 2 0 ± l °C.

Cytological techniques

Embryos were examined chiefly by means of sections. Embryos were fixed ina mixture of formalin, glacial acetic acid, absolute alcohol, and distilled water(6:1:16:30) for 10-24h. The chorion, though permeable to the fixative, isimpermeable to cedar-wood oil and paraffin wax, and was pricked with atungsten needle set in a micromanipulator while the embryos were still in thefixative. The embryos were taken through 50 % cellosolve to a saturatedsolution of eosin in cellosolve, where they were left for 24 h. This stained theembryos bright red so that they could be orientated more easily during embedd-ing. The embryos were cleared in cedar-wood oil and embedded in a mixtureof 56 °C and 58 °C paraffin wax (1:1) containing 3 % beeswax. Sections werecut at 10 /i. In order to facilitate finding the cut sections on the slide, the waxribbons were outlined with an indian-ink mixture developed by Pusey (1939)before dewaxing. Sections were stained with Heidenhain's iron haematoxylin,leucobasic fuchsin counterstained with eosin being substituted in certain cases.

17 EMB 24

260 C. R. BANTOCK

For the histochemical studies the same procedure was used except thatHelly's or Zenker's fixatives were used and that in some cases sections were cutat 5 /.i. The chromosomes of the embryos were examined by means of smears;each embryo was pricked on a slide so that the cytoplasm ran out in a finefilm. The smears were fixed in Zenker's fixative and stained with iron haema-toxylin. The chromosomes of the later stages were examined by means of aceto-orcein squashes, and sometimes by means of sections of material fixed in SanFelice's fluid and stained with crystal violet.

RESULTS

The chromosomes o/Mayetiola

The chromosomes and early cleavage divisions of Mayetiola destructor wereinvestigated by Metcalfe (1935). Although this account is inaccurate in severalrespects it correctly establishes that chromosome elimination occurs at the fifthdivision in this species and also that there is no subsequent elimination ofchromosomes from the presumptive somatic nuclei in male embryos.

The pre-elimination chromosome number

Over 120 embryos were examined by means of smears but it has not beenpossible to count the full chromosome number accurately. This is due to thefact that in division the chromosomes are long and thin, and though thechromosomes of different nuclei never become mixed, the chromosomes ofany one nucleus frequently overlap. The chromosome number was always inthe range 35-45, and in one particular nucleus between 39 and 42 chromo-somes could be counted.

The primordial germ-cell chromosome number

Divisions of the pole cells were examined in post-elimination embryossquashed on slides. The chromosome number was in the same range as in pre-elimination nuclei: 35-45. The larval gonads were also examined but the germcells were too small to be squashed at all satisfactorily. Metcalfe (1935) estimatedthe germ-cell chromosome number entirely by means of 5 fi sections and givesa number of 16 for both sexes. The present work covers a wider range of stagesand indicates that the full chromosome number is a far larger one than Met-calfe's estimation. Many sections of gonads appear to contain germ cells witha small number of chromosomes, but a careful study of the slides shows thatthis is due to the nuclei being cut through and therefore incomplete.

The somatic chromosome number

The female somatic chromosome number is eight; this is characteristic ofmany cecidomyids. A large part of the larval ovary consists of cells of somaticorigin, and when these were observed in division eight chromosomes could be

Chromosome elimination experiments 261counted. There is strong evidence that the male somatic chromosome number isalso eight, unlike the majority of male cecidomyids, which have six chromo-somes in the somatic nuclei. When stages in divisions in embryos between thefifth division and 4 h of development were examined, eight chromosomes couldfrequently be counted. Since Mayetiola frequently but not always producesunisexual batches of eggs, over 200 embryonic smears were examined from overthirty different batches of eggs. At least half of each batch was allowed todevelop to maturity so that some indication of the sex of the embryos could begiven. Even when batches of embryos, some of which had been used for chromo-some study, yielded only male midges, eight chromosomes could be counted inthe somatic prophases.

Cleavage and pole cell formation in Mayetiola

The eggs are cylindrical, approximately 400 fi long and 80 [i in diameter, andare bounded by a transparent chorion. The pattern of cleavage divisions canbe followed clearly in living embryos since the nuclei are surrounded by a densered material (Fig. 1A-D). This material is at first distributed throughout thecytoplasm but gradually accumulates round the synkarion. During the earlyanaphases each red mass can be seen to divide, and between the divisions thecentre of each mass appears to be hollow; this is due to the presence of a largeinterphase nucleus. That the characteristic behaviour of the red masses cor-responded to nuclear divisions was shown by sectioning embryos at definitivestages.

The egg nucleus first appears as a red body lying a third of the distance fromthe anterior end. The first division occurs about 2 h after oviposition and thesubsequent divisions occur at intervals of about 20 min between each anaphase.The first two divisions take place synchronously in all the nuclei but the sub-sequent divisions are asynchronous and take place in a gradient of division, sothat the anterior nuclei start dividing slightly before those situated in moreposterior regions. The primordial germ cells are formed during the fifth division,and in living embryos they can be seen at the posterior end and are character-ized by a reduction in the amount of red material found round the nuclei(Fig. ID).

The cytoplasm is at first irregularly vacuolated, and is slightly more denseround the egg nucleus. This concentration of the cytoplasm round the nucleiincreases during the early divisions and ultimately results in the completeseparation of the cytoplasm from the yolk when the somatic nuclei move to theperiphery to form the blastoderm at 4 h of development. The polar-plasm isindistinguishable from and continuous with the rest of the cytoplasm and ischaracterized by the possession of polar granules (Fig. 1E). These granulesstain very heavily with iron haematoxylin and later become spatially associatedwith the most posterior cleavage nucleus, which enters the polar-plasm. It wasfound that these granules stained red with the Baker modification of the

17-2

262 C. R. BANTOCK

Ant.

25/ 25/*

25/< 25//

H


Altmann-Metzner method for mitochondria. In so far as this procedure is atest for mitochondria the result indicates that at least some or part of the polargranules are mitochondrial in nature, or closely associated with mitochondria.The granules also stained red with the pyronin and methyl green method forRNA. Bradbury's saliva technique (Carleton & Drury, 1957) for the prepara-tion of RN-ase was used as a control, and it was found that the polar granulesthen failed to stain. It may thus be concluded that the polar granules containRNA and probably also mitochondria. Mahowald (1962) has shown that thepolar granules in Drosophila contain RNA, and it is worth pointing out that thegerminal plasm in frogs and toads appears to contain mitochondria and RNA(Bladder, 1958; Czolowska, 1969).

The equatorial plates of the first three divisions are orientated perpendicularto the long axis of the embryo, so that by the end of the third division eightovoid nuclei lie equally spaced in a straight row along the embryo. The appear-ance of a gradient during the third or fourth division is evident; the more

FIGURE 1

A-D. Four living unstained embryos. The cytoplasm is pale orange in colour and ared material surrounds the nuclei.

(A) After the first division. The two nuclei appear as two reddish bodies abouta third of the distance from the anterior end {Ant.). At the posterior end the chorionextends slightly beyond the cytoplasm.(B) After the third division. There are eight nuclei in a single row down the embryo.(C) After the fourth division. There are sixteen nuclei. Two of these are at theposterior end in the polar-plasm.(D) After the fifth division. One of the two pole cells at the posterior end appears asa single vesicle. In the main body of the embryo there are twenty-eight nuclei, onlytwenty-four of which are in focus. These nuclei have undergone chromosomeelimination during the fifth division, but the eliminated chromosomes are not visiblein living embryos.E-I. Longitudinal sections through the posterior end of embryos showing the polargranules and the formation of the primordial germ cell. The sections are stainedwith iron haematoxylin.(E) An uncleaved fertilized egg. The polar granules^.,?.) can be seen at the extremeposterior end.

(F) Before the fourth division. The most posterior nucleus has entered the polar-plasm and the polar granules have begun to form a crescent at the posterior end.A circular constriction has begun to appear round the polar-plasm.(G) The fourth division, showing prophase in the most posterior nucleus.(H) After the fourth division. The circular constriction has been carried further sothat the polar-plasm, the polar granules, and the two posterior nuclei, will beseparated from the rest of the embryo.(I) During the fifth (eliminating) division. A binucleate primordial germ cell is almostcompletely formed, and the polar granules have spread out over the surface of the twonuclei which are not dividing. Part of an elimination metaphase can be seen lyingin the somatic part of the embryo.

264 C. R. BANTOCK

anterior nuclei enter prophase slightly before those near the posterior end ofthe embryo. Immediately before the fourth division the polar granules lying inthe polar-plasm assume the form of a crescent at the posterior end, with themost posterior nucleus lying in the polar-plasm near the granules (Fig. 1F). Atthis time a slight constriction appears round the embryo at the posterior end,so that a polar bud is formed containing the polar-plasm, the polar granules,and a nucleus. During the fourth division all eight nuclei divide with the equa-torial plates parallel to the long axis. Immediately after this division the polargranules spread out and become closely associated with the nuclear membranesof the two nuclei lying in the polar-plasm. During the fifth division these twonuclei do not divide; the fourteen nuclei lying in the somatic part of the embryodivide with chromosome elimination. During this division the posterior trans-verse constriction is carried farther, so that a binucleate primordial germ cell isformed by the time that chromosome elimination is completed in the rest ofthe embryo. A cell membrane appears between the two presumptive germ-linenuclei and thus two pole cells are formed. The main stages in the formation ofthe germ line can be seen in Fig. 1F-I. From the time of the fifth division thepole cells divide less frequently than the somatic nuclei and stop dividing alto-gether by 1\ h of development.

The gradient which appears at the third or fourth division persists during thefifth division so that chromosome elimination takes place as a wave starting atthe anterior end. At the fifth metaphase all the chromosomes lie closely packedon the equatorial plates, which are not noticeably different from those of earlierdivisions. Owing to the extreme rapidity of the process and to the great numberand small size of the chromosomes during anaphase, it is difficult to ascertainthe exact sequence of events during elimination. A small space appears acrossthe equator, suggesting that all the chromatids enter anaphase. However, onlythose chromosomes (S) destined to occupy the presumptive somatic nucleireach the poles; most of the chromosomes (E) return to the equator of eachspindle and coalesce to form a lump of strongly Feulgen-positive material.

The subsequent divisions of the small presumptive somatic nuclei are normal;no elimination of chromosomes has been observed from any nucleus after thefifth division. The ultimate fate of the eliminated chromosomes is uncertain.During embryonic life they break up into smaller pieces, but there is no evidenceeither that this takes place in any regular way or that it is accompanied by anyDNA synthesis. The lumps remain strongly Feulgen-positive and finally dis-appear with the yolk when the young larva starts to feed.

Experiments on chromosome elimination

It is not known whether the nucleus which enters the polar-plasm before thefourth division is predetermined to become the germ-cell nucleus. If, however, itis assumed that mitosis results in initially identical nuclei it follows that it isextremely likely that all of the sixteen nuclei in the Mayetiola embryo before


the fifth division are identical. If this is the case the sudden dichotomy in nuclearbehaviour between those nuclei lying in the somatic part of the embryo and thoselying in the polar-plasm must result from some influence outside the nuclei them-selves. The temporary cessation of mitosis in the two nuclei lying in the polarplasm during the eliminating division in the rest of the embryo, and the charac-teristic behaviour of the polar granules at this time, suggest that the latter maybe concerned with this inhibition of mitosis. The following two types of experi-ment were performed to analyse the function of the polar granules and theirpossible relationship to the non-division of and the non-elimination of chromo-somes from the germ-cell nuclei: (i) the first was an attempt to affect the normalpotentiality of the posterior end by ultraviolet irradiation before any nucleushad migrated into it; (ii) the second was an attempt to alter the normal spatialrelationships of the nuclei, the polar granules, and the polar-plasm, so that aconsistently abnormal situation prevailed at the time of the fifth division. Con-striction and centrifugation procedures were used for this.

Experiments with ultraviolet irradiation

Pieces of embedding wax were used as a base on which to support the embryosduring the experiment. Parallel grooves, 2 mm apart, were made on one surfaceand the embryos transferred to these grooves and arranged with the sameorientation in as straight a row as possible, one in each groove. The embryoswere then covered with a glass coverslip wrapped in silver paper; the positionof the coverslip was adjusted so that only the posterior end of each embryoprojected from under the silver paper. The whole piece of wax was then placedin an ultraviolet beam from a Hanovia 100 W medium-pressure mercury arclamp. The lamp was used without a filter, but with a quartz condenser, andarranged so as to give a downwardly directed beam. [The bulk of the output ofthe lamp was at 2537 A, and it is likely that it was this wavelength that wasoperative in altering the potentialities of the posterior end (J. B. Gurdon,personal communication).] The embryos were placed 12 mm from the source,and were all in the 2- or 4-nuclei stage. Any embryo which had reached the8-nuclei stage by the end of the irradiation was discarded as it was likely thatthe most posterior nucleus in these cases had received at least some irradiation.Three experiments were carried out; the first two were controls.

(i) Seventeen embryos were transferred to wax blocks and completely coveredwith silver-paper-wrapped coverslips. The blocks were irradiated for 12 min.Subsequent development of all the embryos was completely normal.

(ii) Twenty-three embryos were irradiated with no protection whatever fordoses of between 3 and 12 min. In nearly all cases the treatment proved lethalbefore the fourth division.

(iii) Three hundred and twenty embryos were irradiated in batches of aboutsix at a time for 7 min doses, with only the posterior end exposed to the ultra-

266 C. R. BANTOCK

violet beam. The following list gives the stages at which these embryos wereanalysed:

No. ofembryos

Died after irradiation 12Fixed shortly after irradiation 11Fixed during the 5th division 16Fixed at the blastoderm stage (6 h) 15Fixed at later embryonic stages (17 h) 5Analysed at post-embryonic stages 261

Total 320

The first five categories are dealt with here.(1) Twelve embryos died shortly after irradiation. This was probably due to

damage received during transference to and from the wax blocks. It was noticedthat the chorion is particularly fragile in very young embryos (2- or 4-nucleistages).

(2) Eleven embryos were fixed within 5 min of the completion of irradiation.The polar granules in these embryos had lost their clearly granular structure;they appeared to be undergoing disintegration. This was accompanied by adecrease in their staining properties. The polar-plasm itself appeared to beunaffected.

FIGURE 2

Longitudinal sections through normal and experimental embryos. The sections arestained with iron haematoxylin.

(A) A normal 6 h embryo. Small somatic nuclei are becoming surrounded by cellmembranes so that the blastoderm will be formed. At the posterior end are largepole cells containing the polar granules.

(B) A 6 h embryo in which the polar-plasm was irradiated with ultraviolet light at the2-nuclei stage. The polar granules have disappeared and the polar-plasm is occupiedby small nuclei.(C) A 3 h embryo constricted during the third, fourth and fifth divisions so that nonucleus could enter the polar-plasm. All the nuclei divided with chromosomeelimination at the fifth division. Small nuclei then entered the polar-plasm andbecame associated with the polar granules. These nuclei then divided at the slow rateof mitosis characteristic of normal pole cells. The small nuclei of three of these polecells can be seen as pale vesicles completely surrounded by the polar granules. Thearrow indicates the position of the original constriction.(D) A 3£ h embryo centrifuged at the 4-nuclei stage with the polar-plasm placedcentripetally. All the nuclei divided with chromosome elimination at the fifthdivision. During the course of their redistribution after centrifugation small nucleienter the polar-plasm and small pole cells (r.p.c.) are formed. These do not containthe polar granules. The chromatin of the small pole cells is diffusely scattered withinthe nuclear membrane; this is different from the somatic nuclei where the chromatinis concentrated in the centre of the nucleus.

(E) A 4 h embryo centrifuged at the 4-nuclei stage with the polar-plasm placedcentripetally. All the nuclei divided with chromosome elimination at the fifthdivision, and small pole cells (r.p.c.) are forming in a lateral position.


(3) In the sixteen embryos fixed during the fifth division the polar granuleswere either barely visible or had disappeared completely. In no case could therebe observed any association of the granules with the nuclei in the polar-plasm.The irradiation appeared to slow down the formation of primordial germ cells,since although one or two nuclei were always present in the polar-plasm by thetime of the fifth division, there was generally little sign of cytoplasmic con-

268 C. R. BANTOCK

striction. Since the fifth division takes place as a wave of mitosis starting at theanterior end, it has been difficult to determine exactly what happens in thepolar-plasm when the wave of mitosis reaches the posterior end. There is nosign that those nuclei lying in the polar-plasm of an irradiated embryo divideat the fifth division. Some of the sixteen embryos had been fixed just after thisdivision; a variable number of small vesicles containing diffusely scatteredchromatin can be seen in the polar-plasm of these embryos. It is probable thatthese vesicles are derived non-mitotically from the nucleus which entered thepolar-plasm after the third division.

(4) In the fifteen embryos fixed 4 h after irradiation there was no sign of largepole cells (Fig. 2 A). The posterior end was occupied by small cells indistinguish-able from somatic cells in the rest of the blastoderm (Fig. 2B). It is not possibleto say whether the nuclei of these cells are derived from the original primordialgerm cell nucleus or from nuclei of somatic origin which have migrated intothe polar-plasm after the fifth division.

(5) Five irradiated embryos fixed at 17 h of development showed gonadprimordia consisting entirely of small cells with the reduced number of chromo-somes in their nuclei.

Ultraviolet irradiation of the posterior end during the 2- or 4-nuclei stageappears to have the following effects on embryonic development:

(i) The polar granules show a progressive disintegration and reduction instaining power. They do not associate themselves with primordial germ-cellnuclei.

(ii) Constriction of the polar-plasm to form cells is delayed so that it takesplace after the fifth division.

(iii) The two primordial germ-cell nuclei appear to break up into smallervesicles during the fifth division.

(iv) Somatic nuclei appear to replace the abnormal germ-cell nuclei so thata germ line is formed which consists entirely of cells with the reduced chromo-some number in their nuclei.

Experiments involving constriction of embryos

These experiments were designed to prevent any nuclei from entering thepolar-plasm before the fifth division, so that all the nuclei were lying in thesomatic part of the embryo at the time of the fifth division. Embryos with twoor four nuclei were used, and each embryo was treated separately. Fine humanhair was used for the constriction; a hair was weighted at one end with a smallpiece of plasticine and the other end was attached by adhesive tape half-wayalong the long edge of a glass slide. The slide was arranged under a binocularmicroscope so that the hair lay across the slide. One embryo was placed on theslide a few millimetres from the edge and by means of a pin the hair was placedacross the embryo in the required place. The weight of the plasticine tightenedthe hair and caused a constriction to appear across the embryo. After the


fifth division the hair was removed and the embryo returned to its originalshape. One hundred and sixty embryos were constricted; 109 of these survivedand continued to develop after the hair had been removed. In 43 of the 109embryos which survived the constriction the most posterior nucleus passedunder the constriction into the polar-plasm before the fifth division so that theseembryos had the normal distribution of nuclei at the time of the fifth division.It was later ascertained by sections that these embryos possessed pole-cellnuclei with the full chromosome number. In sixty-six embryos the constrictionwas tight enough to prevent the migration of the posterior nucleus into thepolar-plasm at the normal time; in these embryos all sixteen nuclei lay in thesomatic part of the embryo before the fifth division and divided at that division.This was followed by the migration of two posterior nuclei under the constric-tion into the polar-plasm. These sixty-six embryos were analysed at the follow-ing developmental stages:

No. ofembryos

Fixed at the blastoderm stage 12Fixed at later embryonic stages (17 h) 8Analysed at post-embryonic stages 46

Total 66

The first two categories are dealt with here.(1) In the twelve embryos fixed at the blastoderm stage a germ line was present

containing nuclei with the reduced number of chromosomes. Owing to thereduced size of the nuclei occupying the polar-plasm, the polar granules tendto surround them completely instead of forming an irregular crescent (Fig. 2C).The low number of these small pole cells indicates that they were dividing at therate of normal pole cells; that is, much less frequently than the nuclei in thesomatic part of the embryo.

(2) These eight embryos showed the same features as embryos in which theposterior end had been irradiated; gonad primordia were present containingcells with small nuclei.

By preventing the posterior nucleus from entering the polar-plasm at thenormal time all the nuclei undergo elimination at the fifth division. Nuclei withonly the somatic chromosome number then enter the polar-plasm and becomeassociated with the polar granules. These nuclei subsequently divide at the samefrequency as normal pole-cell nuclei, and germ cells are established which arenormal in every way except that their nuclei contain the somatic chromosomenumber only.

Experiments with centrifugation

Since Mayetiola lays its eggs all with the same orientation in longitudinalrows on wheat leaves, and since large numbers of eggs are laid at any one time,

270 C. R. BANTOCK

Direction of centrifugation

Series Number ofembryos

25

Beforecentrifugation

Aftercentrifugation

32 o o o 3>

IV

106

82

30

VI 5. <r

VII 84

VIM 60

Total 470

o o o o o o o„ a o o o o oD

|.V-./.:>| Opaque grey zone

I I Clear yellow zone

Illllll Orange zone

FIGURE 3

Series I-IV are embryos with 2-16 nuclei centrifuged with the posterior end orient-ated centripetally. Series V-VIII are embryos with 2-16 nuclei centrifuged with theposterior end centrifugal. Centrifugation produced dispersal of the contents of theembryo in the form of a density gradient. In all the embryos the cytoplasm becamestratified into three zones. In series I, II, V and VI, the polar granules were dis-persed and could not be located in sections after centrifugation. With series III,IV, VII and VIII (in which the polar granules were associated with a nucleus ornuclei at the moment of centrifugation) it was not possible to separate the posteriornucleus or nuclei from the polar-plasm and polar granules after the associationbetween these had been established (after the third division). The subsequentdevelopment of embryos in series III, IV, VH and VIII was perfectly normal.A germ line was formed with the full chromosome number in the nuclei. Thesubsequent development of embryos in series I, II, V and VI was abnormal. All thenuclei divided at the fifth division and underwent chromosome elimination. Smallpole cells were formed when the somatic nuclei redistributed themselves within theembryo; some of them entered the polar-plasm, which may have been displacedfrom the posterior end. All these embryos died before gastrulation.

Chromosome elimination experiments 111it was found that it was possible to centrifuge large numbers of embryos of thesame age without removing them from the leaves. Each leaf was attached withadhesive tape to a strip of perspex which just fitted into a small centrifuge tube.Embryos were centrifuged at 2-, 4-, 8- or 16-nuclei stages, and were centrifugedin batches of between eight and twenty-five at a time. Eight series of experimentswere carried out, summarized in Fig. 3, involving a total of 470 embryos. It wasfound that the most consistent results were obtained at relatively faster centri-fuge speeds maintained for short periods of time (3-5 min), rather than for longerperiods of centrifugation at slower speeds.

At the faster centrifuge speeds the embryonic cytoplasm became stratifiedinto three zones. This took place irrespective of both the age of the embryo andthe direction of the centrifugal force in relation to the longitudinal axis. At thecentrifugal end of the embryo an orange zone appeared, occupying one-thirdof the embryo; sections showed that this zone consisted of a number of largeglobules, probably of yolk. The middle zone was a clear yellow colour consisting ofwhat appeared to be granular cytoplasm and the centripetal zone was opaque andgrey in colour and consisted of a mass of uniform cytoplasm. In all embryoswith up to four nuclei the polar granules were dispersed so that they did notstain with iron haematoxylin. This dispersal of the polar granules took place atshort periods of centrifugation and also at slower speeds; the granules appearedto be among the first things affected by centrifugation. The polar-plasm isdistinguishable from the rest of the cytoplasm only by the possession of polargranules, and since these granules are dispersed at the onset of centrifugationit is not possible to determine the degree of displacement of the polar-plasm inembryos before the 8-nuclei stage. In embryos with eight or more nuclei thepolar granules were not removed from the polar-plasm, nor were they separatedfrom the nucleus or nuclei lying in the polar-plasm; in these embryos theprimordial germ cell was moved as a single unit.

The degree of displacement of the nuclei was found to be dependent on thedirection and duration of centrifugation, and on the age of the embryo. Up tothe 4-nuclei stage all the nuclei were displaced into the centrifugal orange zone;this was irrespective of the direction of centrifugation. In older embryos thedisplacement was similar except that the primordial germ cells (or cell) weremoved only into the central yellow zone if they were initially centripetal, butthey remained at the posterior end if they were initially centrifugal.

Centrifugation did not alter the course of the nuclear divisions; the nucleisometimes divided during centrifugation. Immediately after centrifugation thenuclei began to redistribute themselves and the cytoplasmic stratifications gradu-ally disappeared. It was noticed that in all cases the gradient of division wasmaintained or reconstituted after centrifugation; the nuclei always divided ina wave which started at the anterior end.

Series I and II. (Fifty-seven embryos with two or four nuclei, centrifuged withthe polar-plasm centripetal.) In these embryos the nuclei did not reach the

272 C. R. BANTOCK

posterior end until between 3 and 4 h after centrifugation; the most posteriornucleus lay about a quarter of the way from the posterior end at the time of thefifth division. This division took place as in normal embryos, as a wave whichstarted at the anterior end. All the nuclei divided at the fifth division, and sectionsshowed that chromosome elimination had occurred throughout. Sections oflater stages showed that cells which resembled small pole cells were formed whenthe nuclei, towards the end of their posterior migration, reached the posteriorend. These can be seen in Fig. 2D. In some embryos of these series what ap-peared to be small pole cells were formed in a lateral position at a short distancefrom the posterior end (Fig. 2E). In these embryos it seems likely that the polar-plasm itself has been moved during the centrifugation and that when small nucleienter it, the polar-plasm becomes constricted to form pole cells before a posteriorposition has been reached.

All the embryos in Series I and II died before gastrulation; the nuclei stoppeddividing at about 4 | h of development. It was thus not possible to follow thefate of these small pole cells beyond the point of their formation.

Series III and IV. (188 embryos with eight or sixteen nuclei, centrifuged withthe polar-plasm centripetal.) In these embryos the position of the primordialgerm cell or cells could be located immediately after centrifugation, since oncea nucleus had come into contact with the polar granules and the polar-plasmit was not possible to destroy the association by centrifugation. Only the pre-sumptive somatic nuclei divided at the fifth division; the germ-line nucleiretained the E chromosomes and divided less frequently than the somaticnuclei. They reached the posterior end in about an hour after centrifugation.In spite of the fact that the embryos in these series were centrifuged for thesame time and speed as those in series I and II where the effect proved lethal, allthese survived and were capable of giving rise to normal adults. In other words,centrifugation of embryos in series III and IV produced no irreversible effecton development.

Series V and VI. (Eighty-one embryos with two or four nuclei, centrifuged withthe polar-plasm centrifugal.) The subsequent development of these embryos wassimilar to that of series I and II. Although the polar-plasm was centrifugal thepolar granules became dispersed and could not be seen in any of the sections.All the nuclei divided at the fifth division and chromosome elimination occurredthroughout. Small pole cells were formed when these nuclei reached theposterior end. As with series I and II, all these embryos died before gastru-lation.

Series VII and VIII. (144 embryos with eight or sixteen nuclei centrifugedwith the polar-plasm centrifugal.) It was not possible to force any more nucleiinto the polar-plasm by centrifugation. The primordial germ cell was relativelyundisturbed by the centrifugation; it was sometimes shifted to a partly lateralposition. The subsequent development of these embryos was normal, as withseries III and IV.

Chromosome elimination experiments 273The following conclusions can be reached concerning centrifugation of

Mayetioia embryos:(1) Centrifugation causes stratification of the cytoplasm into three layers.

This takes place independently of the age of the embryo and of the directionof the centrifugal force.

(2) In embryos centrifuged before a nucleus enters the polar-plasm, the polargranules disappear, probably by becoming dispersed into the somatic part ofthe embryo.

(3) The gradient of division of the nuclei is not changed by centrifugation.(4) If no nucleus is in contact with the polar-plasm and polar granules at the

time of the fifth division, all the nuclei divide with chromosome elimination atthis division. In such embryos small pole cells are formed when nuclei with thesomatic chromosome number enter the polar-plasm. All these embryos diebefore gastrulation.

(5) Centrifugation of embryos in which the primordial germ cell is alreadyformed does not have any permanent effect on development.

Analysis of post-embryonic stages possessing germ-line nuclei with the reducednumber of chromosomes

Three hundred and seven experimental embryos were allowed to develop tomaturity. Forty-six of these were derived from constriction experiments and261 from ultraviolet irradiation experiments. As has already been mentioned,there is an approximately 40% larval mortality at the immediately post-embryonic stage; of the forty-six embryos subjected to constriction procedures,only twenty-six survived to maturity (56% survival), and of the 261 embryossubjected to irradiation only 150 survived to maturity (57 % survival). A controlwith exactly 300 normal embryos gave 193 adults (64% survival), suggestingthat the high mortality rate of experimental embryos is due to factors other thanthose to which they have been subjected experimentally.

Both irradiation and the constriction experiments thus produced someembryos which became adults. When these embryos developed to maturity itwas found that they showed exactly the same degree of abnormality, but it isimportant to appreciate the difference between the two types of germ line. Thegerm line of a constricted embryo differs from the normal germ line only in theabsence of the E chromosomes. The germ line is otherwise unchanged; it hasboth polar granules and an undamaged polar-plasm. On the other hand, in anirradiated embryo the polar granules are destroyed and the polar-plasm itself issufficiently damaged to cause the disintegration of the nuclei which occupy itduring the fifth division. The polar-plasm is later occupied by somatic nuclei,and the polar-plasm is subsequently able to provide an adequate environmentfor the normal division of the nuclei lying in it. The irradiation does not affectthe potentiality of the polar-plasm for forming a germ line, and neither doesthe destruction of the polar granules affect this. The abnormalities found in

274 C. R. BANTOCK

f.c.

Chromosome elimination experiments 275older stages derived from irradiated embryos are the same as those in individualsdeveloped from constricted embryos and evidently result from the absence ofthe E chromosomes from the germ line.

For the greater part of larval life the gonads are identical in male and female,and consist of small groups of cells with nuclei with the reduced chromosomenumber. The gonads lie in the ninth trunk segment and are attached to theMalpighian tubules by fine tracheae.

1. Females. Of the 176 experimental embryos which survived to post-embryonic stages, 159 developed into females (presumably by chance). Fig. 4E

FIGURE 4

Transverse sections through the ovaries of normal and experimental individuals.All the sections are stained with iron haematoxylin except A, which is stained withcrystal violet. A-D, Normal individuals. E-H, Experimental individuals, withovaries lacking the E chromosomes.(A) The ovary in the last larval instar. A large mass of follicle cells (f.c.) can be seenon the left. The larger primordial germ cells, or oogonia (oog.), form a smallergroup to the right.(B) Section through the ovaries in the middle of the pupal instar. The follicles con-tain groups of oogonia and are attached to the inner end of the oviduct (ov.).(C) Transverse section through the abdomen of a late pupa, showing the twoovaries much enlarged due to the formation of ooplasm {pop.) in the follicles (/.).Each follicle contains a group of nurse cells (n.c.) with deeply staining cytoplasm.(D) The same individual as C, showing the follicles composed of follicle cells (/c.)containing the nurse cells (n.c.) and ooplasm. The ooplasm of the oocyte in eachfollicle is lightly stained and the nurse cells have large spherical nuclei with deeplystaining irregular chromosomes.(E) The ovary in a late larva derived from an embryo in which the polar-plasm wasultraviolet irradiated at the 2-nuclei stage. The ovary consists entirely of small cells(r.). The oviduct (ov.) can be seen.(F) Transverse section through the abdomen in the middle of the pupal instar. Thisindividual was derived from an embryo in which the polar-plasm had been ultra-violet-irradiated at the 2-nuclei stage. The follicles (/.) are attached to the oviduct(ov.) and have remained small at the stage when they would normally have enlargedand filled the abdomen. The follicles contain groups of cells with only the somaticnumber of chromosomes. The large abdominal space was originally filled with redpigment; in normal individuals this pigment is deposited in the ooplasm of thedeveloping oocytes.

(G) Transverse section through part of the ovary towards the end of the pupal instar.This individual was derived from an embryo in which the posterior end had beenconstricted at the 2-nuclei stage so that no nuclei could enter the polar-plasm. Thefollicles (f.c.) are enlarged and contain groups of closely packed cells (r.n.c.) withonly the somatic chromosome number. There are no oocytes and hence no ooplasm.ov. = oviduct.(H) Transverse section of part of the ovary of an adult derived from an embryo whichwas constricted at the 2-nuclei stage. The groups of small cells (r.n.c.) in the follicleshave broken down and most of these cells have been passed into the oviduct (ov.),and now lie in the vagina.l8 EMB 24

276 C. R. BANTOCK

shows the appearance of the ovary in a larva just before pupation; the larva isderived from an embryo subjected to irradiation. The ovary consists entirely ofcells with the somatic number of chromosomes; there are none of the oogoniawhich are found in the normal ovary (Fig. 4 A). The greater part of the normalovary consists of follicle cells of somatic origin. These later form follicles whichsurround groups of oogonia derived from the primordial germ cells, and alsoform the inner end of the oviduct (Fig. 4B). All but one of the oogonia in eachfollicle form large nurse cells with deeply staining cytoplasm (Fig. 4C). Oneoogonial cell in each follicle becomes an oocyte; each of these subsequentlyenlarges so that the whole ovary fills the abdomen.

In the experimental females follicles are formed but these contain only groupsof cells with nuclei with the somatic chromosome number. These cells are pre-sumably derived from the small germ cells formed in the embryo. It is duringthe pupal instar that the effects of the absence of the E chromosomes becomeapparent. The cells in the follicles show no differentiation into nurse cells andoocytes. Even though there are no oocytes in the follicles, these enlarge so thattheir walls become folded and spaces appear between the follicle walls and thegroups of cells in each follicle (Fig. 4G). Oocyte enlargement in normal pupaeis accompanied by the appearance of a secretion which stains strongly withiron haematoxylin and which appears to pass from the nurse cells into theooplasm in each follicle. The whole of the ovary becomes red in colour due tothe appearance of red pigment which is deposited in the ooplasm and which laterpersists round the nuclei of the developing embryo. The deeply staining secretionis absent from the ovaries of experimental embryos. The red pigment, however,does appear, so that the pupal abdomen becomes swollen and red in colour.It was found that the pigment, instead of being deposited in the follicles, wassecreted in the large abdominal space (Fig. 4F) which in the normal pupa wouldhave been occupied by the enlarged ovary.

Towards the end of pupal life the remainder of the reproductive system de-velops. This consists of the egg-laying apparatus, accessory glands and sperma-theca, and all were normal in the experimental individuals. It was found that inlate experimental pupae the small cells occupying the follicles began to separate(Fig. 4H) and were eventually discharged irregularly into the oviduct afteremergence of the imago. Later they were passed as far back as the vagina, bywhich time they had broken up and their nuclei had assumed the form of deeplyFeulgen-positive globules. It was noticed that the cells began to be dischargedinto the oviducts at the time that the oocytes are released into the reproductivetract of a normal female individual, and that this was continued during thetime that normal egg-laying would have taken place.

Fifty-four female midges were obtained from the experimental embryoswhich survived. These adults appeared to be perfectly normal except in repro-ductive capacity. They were capable of mating with normal and experimentalmales and after mating attempted to lay eggs by walking up wheat leaves and

Chromosome elimination experiments 277extending their ovipositors. Occasionally small droplets of colourless fluid weredeposited.

The absence of the E chromosomes from the germ line of the female is thusassociated with the following developmental abnormalities:

(a) Only the germ cells are affected. The rest of the body, including thesomatically derived part of the ovary and the rest of the reproductive system,is normal.

(b) Ovarian follicles are formed but these contain only small cells which showno differentiation into oocytes and nurse cells.

(c) The oocyte growth stage is deficient and the midges are sterile; sterility ismanifest from the moment that gametes would have begun to develop had theindividuals been normal.

(d) In the adult female midge the cells occupying the follicles become free andare discharged into the oviducts. They accumulate in the ovipositor, by whichtime they have broken down and disintegrated into Feulgen-positive globules.

(<?) The behaviour of the female midges is unaffected. They mate and attemptto lay eggs.

2. Males. Of the 154 experimental embryos which survived, only seventeendeveloped into males and so information concerning the development of ab-normal testes is based on small numbers. Up to the middle of the last larvalinstar the testes in the normal male consist of a spherical mass of primordialgerm cells, surrounded by a thin wall of somatic cells. Early in the pupal instarsome of the primordial germ cells become large 'nutritive' cells; the remainderare spermatogonia (Fig. 5 A). The spermatogonia develop by two divisions intospermatozoa. The first division involves unequal cytokinesis and results in theformation of small inter-kinetic cells with four chromosomes and an equalnumber of residual cells containing the remainder of the chromosomes pos-sessed by the original spermatogonia. The second division concerns only theinter-kinetic cells and results in the formation of spermatids. These developtails before the end of pupal life and the resultant spermatozoa are passed byvasa deferentia to a seminal vesicle lying near the posterior end of the abdomen.The nucleus of the spermatozoon is divisible into two parts (Fig. 5B, C).

During the larval instars and the first part of the pupal instar a testis lackingthe E chromosomes consists of a mass of small cells surrounded by a wall ofsomatic cells (Fig. 5 D). The central cells are presumably derived from the smallgerm cells formed in the embryo, and they fail to show any sign of differentiationinto 'nutritive' cells and spermatogonia. During the pupal instar the wall ofeach testis becomes more distinct and the central cells become separated fromeach other. The nuclei of these cells do not show any single consistent appear-ance; sometimes they are divided into two parts, as in a normal spermatidnucleus, but more usually they are irregular in shape and size (Fig. 5E). Theyare approximately twice the size of normal spermatid nuclei and the cytoplasmof these cells is sometimes elongated into tails, but this is not a consistent

18-2

278 C. R. BANTOCK

feature. It appears then that cells with the somatic chromosome number in themale gonad attempt to form gametes in the sense that some of them come toresemble spermatids. This appears to take place without division.

The rest of the reproductive system develops towards the end of the pupalinstar and is completely normal in experimental males. Just before emergenceof the male midge the testis tends to become emptied of the cells lying in it;these are passed through the vas deferens and sometimes a few of them reachthe anterior end of the seminal vesicles. By this time the cells have disintegratedand their nuclei have assumed the form of Feulgen-positive globules (Fig. 5F).

Chromosome elimination experiments 279Experimental males were capable of mating with both normal and experi-

mental females; the tendency to mate was not diminished in either sex. Inmatings between normal individuals, spermatozoa are passed to the spermathecaof the female. When a sterile male mated there was no transmission of gametesor of cells of any kind; the spermatheca was quite empty after such a mating.

The absence of E chromosomes from the male germ line is associated withthe following abnormalities:

(a) Only the gonads are affected. The rest of the body, including the rest ofthe reproductive system, is normal.

(b) The testes fail to enlarge at the usual time during the pupal instar, andlarge 'nutritive' cells are not formed.

(c) Some of the small cells occupying the testes assume a slight resemblanceto spermatids. This takes place apparently without division. The nuclei becomecompact and occasionally divided into two parts. They are larger than normalspermatid nuclei. The cytoplasm of some of these cells tends to become elong-ated into a tail-like structure.

id) Functional gametes are not formed, and the midges are sterile. The cellsin the testes are passed into the vasa deferentia, where they begin to dis-integrate ; a few deeply staining dead nuclei are passed as far back as the seminalvesicles.

(<?) The mating behaviour of the male midges is normal.

FIGURE 5

Sections through the testes in normal and experimental individuals. All the sectionsare stained with iron haematoxylin. A-C, Normal individuals. D-F, Experimentalindividuals, with testes lacking the E chromosomes.

(A) The testis early in the pupal instar. Four large 'nutritive' cells (nut.c.) can be seen,with the primordial germ cells, or spermatogonia (s.), arranged loosely round them.

(B) The testis (V.) in a pupa just before emergence. Most of the spermatozoa (sp.)have already passed to the seminal vesicles, but some can be seen entering the vasdeferens (vas.). The residual cells (res.) and the 'nutritive' cells remain in the testis.

(C) Part of a seminal vesicle in a pupa just before emergence. Spermatozoa (sp.)can be seen entering the seminal vesicle (s.v.) at the anterior end. Spermatozoa canalso be seen in the vas deferens (vas.). The nucleus of each spermatozoon is dividedinto two parts.

(D) The testes in a mid-pupa derived from an embryo which was constricted at the2-nuclei stage. Each testis consists of a mass of small cells (r.).

(E) A testis (/.) towards the end of the pupal instar of an individual derived from anembryo in which the polar-plasm was ultraviolet-irradiated at the 2-nuclei stage.The testis is sac-like in form and leads into the vas deferens (vas.). It contains cells thenuclei of some of which have assumed a resemblance to spermatid nuclei (lsp.').

(F) Part of the seminal vesicle in an adult derived from an embryo in which thepolar-plasm was ultraviolet-irradiated at the 2-nuclei stage. The anterior end containsFeulgen-positive particles (' sp.') derived from the cells originally occupying the testis.

280 C. R. BANTOCK

DISCUSSION

The observations on the development of normal and experimental indivi-duals of Mayetiola establish that:

(1) A cleavage gradient exists during the first 4 h of development; the nucleitowards the anterior end of the embryo enter prophase slightly before thosenearer the posterior end of the embryo.

(2) Chromosome elimination occurs at the fifth division when fourteennuclei divide; each nucleus loses between 31 and 34 chromosomes. The elimin-ated chromosomes are called the E chromosomes.

(3) The somatic nuclei contain eight chromosomes, the S chromosomes.(4) The moment of the elimination of chromosomes from the presumptive

somatic nuclei cannot be experimentally altered; it always occurs at the fifthdivision.

(5) The germ line develops from pole cells which are formed at the posteriorend of the embryo. The entry of nuclei into the polar-plasm at the posterior endis marked by a reduction in the frequency of mitosis of these nuclei which retainthe E chromosomes during their divisions.

(6) The two germ-line nuclei do not divide during the fifth division of thepresumptive somatic nuclei.

(7) The posterior end of the embryo contains polar granules which becomeassociated with the membranes of the germ-line nuclei during the fifth division.

(8) The granules cannot be regenerated if they are destroyed during cleavage.(9) The polar granules consist at least in part of RNA and probably mito-

chondria.(10) The polar granules are not necessary for pole cell formation.(11) The E chromosomes are not necessary for the formation of pole cells.(12) The E chromosomes are necessary for the formation of gametes.(13) All the nuclei are capable of undergoing chromosome elimination at the

fifth division. This occurs if nuclei are prevented from entering the polar-plasmbefore the division.

The various aspects of the work will be discussed in turn.

The cause of chromosome elimination

The present work does not elucidate the causes of chromosome elimination,but it is useful to consider the past approaches to this phenomenon, and also tothe comparable case of Ascaris (Nematoda), where parts of chromosomes arelost from the somatic nuclei during cleavage (chromosome diminution). Thefirst experimental work on Ascaris was performed by Boveri (1910). On thebasis of abnormal cleavages after centrifugation of eggs and embryos of Ascaris,Boveri came to the conclusion that diminution was caused by cytoplasmiczones situated perpendicular to the animal/vegetal axis, and that these zonescaused diminution in the somatic part of the embryo and prevented it from


occurring in the germ-line nucleus. With respect to chromosome elimination inMiastor, Kraczkiewicz (1936) suggested that there were cytoplasmic zoneswhich were responsible for changing the state of the spindle and which couldcause elimination. He considered that the existence of a gradient of eliminationwas evidence of the presence of cytoplasmic zones. Du Bois (1933), workingwith Sciara, suggested that there were concentrically arranged zones in theembryo, and that elimination took place when the nuclei lay in the appropriatezones. The present work on Mayetiola shows that chromosome elimination cantake place anywhere in the somatic part of the embryo and does not appear tobe linked to the existence of cytoplasmic zones or centres in the embryo.Geyer-Duszynska (1959) showed that in Wachtliella chromosome eliminationcan take place anywhere in the cytoplasm, and even in the polar-plasm providedthat the polar granules have been removed from this area. The constrictionexperiments show that in Mayetiola chromosome elimination takes place at thefifth division irrespective of the number of nuclei lying in the somatic part ofthe embryo. It is thus likely that the entire embryonic cytoplasm allows elimina-tion to take place in it.

The immediate causes of elimination are unknown. All other authors (White,1950; Geyer-Duszynska, 1959; Nicklas, 1959, 1960) have been of the opinionthat the E chromatids are attached normally to the spindle fibres during ana-phase. In Monarthropalpus the ii chromatids do not separate and White (1950)suggests that the E chromatids are held together by some matrix and that thiswas the immediate cause of elimination. In both Mayetiola and Wachtliella theE chromatids separate completely but fail to continue anaphase, and Geyer-Duszynska (1959) suggests that functional defects in the centromeres of theE chromatids were responsible for causing elimination. By means of the ultra-violet irradiation of small areas of cytoplasm round the nuclei before elimina-tion, Geyer-Duszynska showed that in the absence of a spindle the is chromatidswere capable of completing anaphase during the division in which they wouldnever normally have reached the poles. Since a normal spindle is thus necessaryfor chromosome elimination it is likely that elimination is due to some activefactor which is centred in or around the spindle. At present the origin and natureof this factor is unknown.

The causes of the retention of the E chromosomes by the germ-line nuclei

The formation of the binucleate primordial germ cell (pole cell) is marked bytwo events. The first is an increase in the time between each mitosis of the germ-line nuclei and the second is that the germ-line nuclei retain the E chromosomesduring their divisions. A similar situation occurs in Monarthropalpus, Wachtliellaand Miastor.

If it is accepted that elimination is caused by the action of some factor roundthe cleavage nuclei it follows that this factor must either be absent from thepolar-plasm, or that if it is present, its action must be inhibited in some way if

282 C. R. BANTOCK

the germ-line nuclei are to retain the E chromosomes. Since in Mayetiola thepolar granules become associated with the nuclear membranes from the time ofthe fifth division, when the germ-line nuclei do not divide, up to 14 hof development, during which time the germ-line cells are dividing only in-frequently, it is likely that the granules are in some way concerned with both theretention of the E chromosomes when the germ-line nuclei divide and with thelow frequency of these divisions. The constriction experiments on Mayetiolaindicate that the retention of the E chromosomes is caused by a posterior factor.When a cleavage nucleus is prevented from entering the polar-plasm before thefourth division it divides with loss of chromosomes at the time when it wouldnormally have retained them and not divided. Nuclei with the reduced chromo-some number then enter the polar-plasm, become associated with the polargranules, and subsequently divide less frequently than somatic nuclei. Geyer-Duszynska (1959) found that in Wachtliella division with elimination took placein the polar-plasm provided that the polar granules had been removed from thearea. She also found that in some of the embryos the polar granules were movedby centrifugation into the somatic part of the embryo as a single mass. If thismass came into contact with a nucleus with the full chromosome number thenucleus divided less frequently and retained the E chromosomes. This suggeststhat the polar granules are directly concerned both with the non-division of thegerm-line nuclei during the time that chromosome elimination occurs in therest of the embryo, and also with the onset of the lower frequency of mitosis ofthe germ-line nuclei. In Mayetiola the polar granules become displaced and aredispersed very easily by centrifugation. This is unusual since it has been foundthat in other Diptera the polar granules are not moved very easily (Howland,1941; Nicklas, 1959). Because the polar granules are dispersed in Mayetiola anyeffect they may have had is lost and all the nuclei divide with chromosomeelimination at the fifth division in spite of the presence of dispersed polargranules in the somatic part of the embryo. Nuclei with the reduced chromosomenumber sometimes enter the polar-plasm after centrifugation, but since theseembryos unfortunately die it is not possible to ascertain whether the small polecells that are formed, lacking both polar granules and E chromosomes, divideat a different rate from the somatic nuclei.

It is interesting to speculate as to how the polar granules carry out theirapparent function of causing the retention of the £ chromosomes. The way theydo this must depend on the original source of the factor causing elimination.If this factor is already present in the cytoplasm the granules could prevent itfrom coming into contact with the chromosomes by preventing it from enteringthe germ-line nuclei. If on the other hand the elimination inducer is produced bythe cleavage nuclei themselves then the polar granules could function by pre-venting such a factor from leaving the germ-cell nuclei. In either case it isprobable that the granules function by blocking the activity and penetration orrelease of an elimination inducer. Or in other words the granules interfere with


some nucleo-cytoplasmic interaction which would otherwise result in the appear-ance of irreversible changes to the nuclei in the form of loss of chromosomes.

Cleavage gradients and chromosome elimination

Dipteran development is characterized by the presence of a cleavage gradient(Krause & Sander, 1962); generally this is centred at the point of formation ofthe first nucleus and causes the cleavage nuclei nearest this point to enter pro-phase slightly before those farther away. A cleavage gradient is present inCecidomyidae and it follows that elimination takes place in a gradient. Kracz-kiewicz (1936) considered that the presence of a gradient in cecidomyid de-velopment was evidence of the presence of cytoplasmic zones which wereresponsible for causing chromosome elimination. However, since a cleavagegradient exists in insects which do not undergo elimination, and since thegradient operates at divisions other than those at which elimination occurs, itis likely that the two phenomena are unrelated. It is unlikely that the polargranules (which are associated with a lower frequency of mitosis of the germ-line nuclei) are responsible for the appearance of a cleavage gradient since thegradient exists after centrifugation even when the polar granules are dispersedor moved in a mass into the somatic part of the embryo.

The function of the E chromosomes

After their elimination from the presumptive somatic nuclei the E chromo-somes form strongly Feulgen-positive lumps lying in the somatic part of theembryo. These lumps break up but do not undergo mitosis; they eventuallydisappear when the yolk is digested completely. Nicklas (1959) has shown byFeulgen cytophotometry that DNA is not synthesized in the E chromosomesafter their elimination in Miastor and it is likely that this is true for all Ceci-domyidae. The E chromosomes are thus unlikely to contribute to the develop-ment of the soma after their elimination. On the other hand, the present workon Mayetiola shows that the E chromosomes are concerned exclusively withgamete formation and confirms the work of Geyer-Duszynska (1966) on Wacht-liella persicariae. Mayetiola individuals lacking the E chromosomes from theirgerm-line nuclei are normal in every respect other than in gamete formation.In females the effects of the absence of the E chromosomes become manifestwhen oocytes should be formed; there is a total failure to form both ooplasmand some of the accompanying secretions. Painter (1966) suggests that theE chromosomes serve to increase the polysome-forming capacity of the nursecells; in Drosophila the nurse cells show a series of endomitotic divisions whichare associated with an increased yield of nucleolar material. It is not knownwhether the E chromosomes of the nurse cells in Cecidomyidae are concernedwith polysome formation, but the fact that the effects of the absence of theE chromosomes become manifest at the time that ooplasm would be expectedto develop suggests that this may be so. In males the abnormalities associated

284 C. R. BANTOCK

with the absence of E chromosomes are less drastic since some cells are formedwhich resemble gametes. These cells are unable to fertilize normal eggs.

The significance of chromosome elimination

An understanding of the significance of chromosome elimination must berelated to that of the evolution of the cytogenetic system found in the Cecido-myidae. As White (1950) points out, on morphological evidence the Cecido-myidae belong to the suborder Nematocera of the Diptera, and most probablyform a natural group together with the Mycetophilidae and the Sciaridae. TheMycetophilidae are considered to be the most primitive and in this family thegerm line and somatic chromosome number are the same. In those Sciaridaewhich have been analysed cytologically the elimination of two or three auto-somal chromosomes takes place from the somatic nuclei during cleavage. In theCecidomyidae the full chromosome number is very much larger than it is in theSciaridae and in some but not in all is a multiple of the somatic number.Nicklas (1959) has shown by measurements of the centromere positions and therelative lengths of the chromosomes that half the germ-line chromosome numberin Miastor cannot be homologous with the somatic number. On the other handthe possibility of tetraploidy of the haploid somatic number is not excluded.Since the E chromosomes do not form bivalents during meiosis it is likely thatthey form a genetically distinct chromosomal group, and since in the moreprimitive Mycetophilidae the somatic and germ-line nuclei have the samechromosome number it is likely that the large chromosome number inCecidomyidae is of a secondary nature and has arisen by an increase of anoriginally smaller number. In other words, a system seems to have arisenwhereby the genes necessary for gametogenesis have accumulated in particularchromosomes which are for some reason eliminated from the somatic nuclei.There is no explanation as to why this separation should have occurred betweensomatic and germ-line genes, and there is no explanation as to the origin of theextra chromosomes. It is also not obvious why the necessary rearrangementwithin the genotype which would give the extra chromosomes their particularfunctions in the germ line should have been selected for. Nicklas (1960) con-siders that chromosome elimination concerns the preservation of a certainnucleo-cytoplasmic ratio based on the volume of the nuclei relative to thevolume of cytoplasm surrounding them. This interpretation could apply to theCecidomyidae and the Orthocladiinae, but hardly to the Sciaridae, where theeliminated chromosomes are so few in number.

In conclusion it can be said that chromosome elimination represents anextreme example of the loss of totipotency of somatic nuclei during develop-ment and must be regarded as an extreme specialization not having any obviousrelation to the development of animals which do not show similar changes.


RESUME

Experiences sur des eliminations chromosomiques chez CecidomydeMayetiola destructor

La segmentation chez Cecidomyidae (Diptera) se caracterise par des eliminations dechromosomes dans les noyaux somatiques presomptifs. La formulechromosomiale completeest conservee dans les noyaux de la Jignee germinale.

Le deroulement de la segmentation est decrit chez Mayetiola destructor (Say). Apres la4eme division, deux noyaux se trouvent dans le plasme polaire posterieur et s'associent auxgranules polaires, tandis que 14 noyaux se situent dans le reste du cytoplasme. Tous les noyeauxpossedent a peu pres 40 chromosomes. Pendant la 5eme division, les noyaux posterieurs nese divisent pas et le plasme polaire se pince pour former les cellules germinales primordiales(cellules polaires). Les 14 autres noyaux se divisent et perdent a peu pres 32 chromosomesde sorte que les 28 noyaux ainsi formes ne possedent que huit chromosomes. Ce sont lales noyaux somatiques. Au cours des divisions subsequentes, les noyaux des cellulespolaires gardent la formule chromosomiale complete; ces divisions sont moins frequentesque celles des cellules somatiques.

II a ete fait des experiences sur les stades embryonnaires precoces arm d'elucider les pro-prietes de la partie posterieure de Poeuf au cours de la periode d'elimination chromosomiquea partir des noyaux somatiques. On a utilise l'irradiation aux rayons ultra-violets, la con-striction et la centrifugation.

Les granules polaires jouent un role dans l'absence de division des cellules germinales aucours du 5eme cycle mitotique car, lorsque ces granules sont disperses par la centrifugationou lorsque les noyaux sont empeches de venir a leur contact, avant la 5eme division, a lasuite d'une constriction, tous les noyaux se divisent avec elimination chromosomique aucours de cette 5eme division.

Des individus ayant une lignee germinale avec seulement huit chromosomes ont ete menesa maturite. Les anomalies n'ont ete observees que dans la lignee germinale et ont ete identiquesquelle qu'ait ete la technique utilisee pour produire cette deficience de la lignee germinale.Un ovaire contenant des cellules germinales a huit chromosomes seulement est incapable deconstituer des oocytes ni des cellules nourricieres. Un testicule contenant des cellules germi-nales a huit chromosomes est incapable de former des spermatocytes mais bien des cellulesqui ont quelque ressemblance avec les gametes. Ces males et femelles experimentaux etaientegalement steriles.

Les resultats sont discutes dans le cadre d'autres travaux experimentaux sur les Ceci-domydes et on a pu arriver aux conclusions suivantes: (a) les granules polaires sont respons-ables d'empecher la perte irreversible des noyaux de cellules germinales et les empechent dese diviser au cours du 5eme cycle; (b) les chromosomes retenus normalement dans la ligneegerminale sont requis pour la gametogenese, en particulier 1'oogenese.

La signification de l'elimination chromosomique est discutee.

This work was carried out in the Department of Zoology of Oxford University during thetenure of a Christopher Welch Scholarship. I am grateful to many members of the Depart-ment for the help I was given, and in particular to Professor M. Fischberg and to Dr J. B.Gurdon for their advice, supervision, and many helpful suggestions. I would like to thankMr R. Haveley and Miss L. Hartwell of the Department of Biology and Geology of theNorthern Polytechnic for their assistance with the Plates.

REFERENCES

BANTOCK, C. (1961). Chromosome elimination in Cecidomyidae. Nature, Lond. 190, 466-467.BLACKLER, A. W. (1958). Contribution to the study of germ cells in the Anura. /. Embryol.

exp. Morph. 6, 491-503.BOVERI, T. (1910). Uber der Teilung Centrifugierter Eier von Ascaris megalocephala. Arch.

EntwMech. Org. 30, 101-145.

286 C R. BANTOCK

CARLETON, H. M. & DRURY, R. A. B. (1957). Histochemical Technique. Oxford UniversityPress.

CZOLOWSKA, R. (1969). Observations on the origin of the 'germinal cytoplasm' in Xenopuslaevis. J. Embryo]. exp. Morph. 22, 229-251.

Du Bois, A. M. (1933). Chromosome behaviour during cleavage in the eggs of Sciaracoprophila (Diptera) in relation to the problem of sex determination. Z. Zellforsch.mikrosk. Anat. 19, 595-614.

GEIGY, R. (1931). Action de l'ultra-violet sur le pole germinal dans l'oeuf de Drosophila melano-gaster. Revue suisse Zool. 38, 187-288.

GEYER-DUSZYNSKA, I. (1959). Experimental research on chromosome elimination in Ceci-domyidae (Diptera). / . exp. Zool. 141, 391-448.

GEYER-DUSZYNSKA, I. (1961). Spindle and chromosome behaviour after partial embryoirradiation. Chromosoma 12, 233-247.

GEYER-DUSZYNSKA, I. (1966). Genetic factors in oogenesis and spermatogenesis in Ceci-domyidae. In Chromosomes Today, vol. 1 (ed. C. D. Darlington & K. R. Lewis), pp. 174-178. Edinburgh: Oliver and Boyd.

HOWLAND, R. B. (1941). Structure and development of centrifuged eggs and early embryosof Drosophila melanogaster. Proc. Am. phil. Soc. 84, 605-616.

KAHLE, W. (1908). Die Paedogenesis der Cecidomyiden. Zoologica, Stuttg. 21, 1-80.KRACZKIEWICZ, Z. (1936). Etudes cytologiques sur l'oogenese et la diminution de la chroma-

tine dans les larves paedogenetiques de Miastor metraloas (Meinert) (Diptera). Folia morph.6, 1-37.

KRAUSE, G. & SANDER, K. (1962). Ooplasmic reaction systems in insect embryogenesis. Adv.Morphog. 2, 259-303.

MAHOWALD, A. P. (1962). Fine structure of pole cells and polar granules in Drosophilamelanogaster. J. exp. Zool. 151, 201-207.

METCALFE, M. (1935). The germ cell cycle in Phytophaga destructor, Say. Q. Jl Microsc. Sci.11, 585-603.

NICKLAS, R. B. (1959). An experimental and descriptive study of chromosome elimination inMiastor spec. Chromosoma 10, 301-336.

NICKLAS, R. B. (1960). The chromosome cycle of a primitive cecidomyid—Mycophila speyeri.Chromosoma 11, 403-419.

PAINTER, T. S. (1966). The role of the ^-chromosomes in Cecidomyidae. Proc. natn. Acad.Sci., U.S.A. 56, 853-855.

PUSEY, H. K. (1939). Methods of reconstruction from microscopic sections. Jl R. microsc.Soc. 59, 232-244.

WHITE, M. J. D. (1950). Cytological studies on Gall Midges (Cecidomyidae). Univ. TexasPubl. No. 5007.

(Manuscript received 6 November 1969)

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Experiments on chromosome elimination in the gall midge, Mayetiola