/. Embryo!, exp. Morph. Vol. 24, 2, pp. 257-286, 1970 257 Printed in Great Britain Experiments on chromosome elimination in the gall midge, Mayetiola destructor ByC. R. BANTOCK1 From the Department of Zoology, Oxford University SUMMARY Cleavage in Cecidomyidae (Diptera) is characterized by the elimination of chromosomes from presumptive somatic nuclei. The full chromosome complement is kept by the germ-line nuclei. The course of cleavage in Mayetiola destructor (Say) is described. After the fourth division two nuclei lie in the posterior polar-plasm and become associated with polar granules, and fourteen 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 becomes constricted to form primordial germ cells (pole cells). The remaining fourteen nuclei divide and lose about thirty-two chromosomes so that twenty-eight nuclei are formed containing only eight chromosomes. These are the presumptive somatic nuclei. During subsequent divisions the pole cell nuclei retain the full chromosome number; these divisions occur less frequently than those of the somatic nuclei. Experiments were performed on early embryonic stages to elucidate the properties of the posterior end during the time that chromosome elimination was taking place from the pre- sumptive somatic nuclei. Ultraviolet irradiation, constriction, and centrifugation techniques were used. The polar granules are concerned with the non-division of the germ-cell nuclei during the fifth division, since if the granules are dispersed by centrifugation, or if nuclei are prevented by constriction from coming into contact with them before the fifth division, all the nuclei divide with chromosome elimination at this division. With each technique it is possible to obtain embryos possessing germ cells with only eight chromosomes in their nuclei. Individuals possessing germ-line nuclei with only eight chromosomes were allowed to develop to maturity. Abnormalities were confined to the germ cells only and were the same regardless of which technique had been used to produce the deficient germ line. An ovary containing germ-cell nuclei with only eight chromosomes is unable to form both oocytes and nurse cells. A testis containing germ-cell nuclei with only eight chromosomes is unable to form spermatocytes but cells which come to resemble gametes are formed. Experimental males and females are both sterile. The results are discussed in relation to other experimental work on Cecidomyidae and the following main conclusions are reached: (a) the polar granules are responsible for preventing an irreversible loss of chromosomes from the germ-cell nuclei by preventing the mitosis of these nuclei during the fifth division; (b) the chromosomes normally retained in the germ line are 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 is well established that experimental procedures which interfere with the normal association between the germ-line nuclei and the cytoplasm these occupy can result in sterility (e.g. Geigy, 1931). In the Cecidomyidae (Diptera) germ cells form as a result of one or more of the posterior cleavage nuclei migrating into the cytoplasm at the posterior end of the embryo (polar-plasm) and the polar-plasm becoming constricted off to form germ cells (pole cells). There is no evidence that in this family of insects the 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. These enter mitosis and a large but specific number of the chromosomes remains at the equator at anaphase and fails to enter the daughter nuclei. This is known as chromosome elimination and was first observed in Miastor by Kahle (1908). The chromosomes which are eliminated are designated E chromosomes, and the remainder S chromosomes. Somatic nuclei have only the S number and the primordial 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 this difference between the elimination pattern of the two sexes is important in sex determination in Cecidomyidae. The formation of pole cells in Cecidomyidae is marked not only by the elimination of chromosomes from the presumptive somatic nuclei but also by the onset of a lower frequency of mitosis of the germ-line nuclei. Chromosome elimination is a particularly clear example of nuclear differentiation at the genetic 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 the causes of the retention of the E chromosomes by the germ-line cells, on the factors 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 species continuously through the year. MATERIAL AND METHODS The eggs of Mayetiola are laid in longitudinal rows on the leaves of wheat plants in the 2-leaf stage. The eggs are deposited always with the posterior end of each nearest the axil of the leaf. Generally the eggs from any one female will develop into adults of only one sex. The eggs hatch in 3-5 days and the larvae migrate to a position under the ligule. This position is maintained throughout larval 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 months in England. Under natural conditions there are three generations a year, the last generation overwintering in the puparia. Emergence takes place early in the 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 Petri dishes containing moist filter paper, but it was not possible to prevent emergence once 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 at 20 °C. Adults emerged in 3—14 days. Those adults required for maintaining the stocks were transferred to potted Peko wheat plants in the 2-leaf stage. The plants were enclosed by glass cylinders closed with muslin at the top. There is a mortality rate of approximately 40% in young larvae before they become established under the ligule; they are particularly susceptible to desiccation. Humidity was maintained in the cylinders by covering the tops with wet cloths when oviposition was completed. The cylinders were removed and the plants covered with muslin bags when all the eggs had disappeared from the leaves. When eggs were required for experimental purposes short pieces of leaf were placed in glass tubes containing mated females. Embryos and young larvae were handled with silver-plated entomological pins set in matchsticks. Post-experimental embryos required for analysis in later stages in the life-cycle were transferred to a drop of water in the axil of a leaf, from which the larvae were able to take up their normal position. All experimental embryos were kept at20±l °C. Cytological techniques Embryos were examined chiefly by means of sections. Embryos were fixed in a 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, is impermeable to cedar-wood oil and paraffin wax, and was pricked with a tungsten needle set in a micromanipulator while the embryos were still in the fixative. The embryos were taken through 50 % cellosolve to a saturated solution of eosin in cellosolve, where they were left for 24 h. This stained the embryos 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 mixture of 56 °C and 58 °C paraffin wax (1:1) containing 3 % beeswax. Sections were cut at 10 /i. In order to facilitate finding the cut sections on the slide, the wax ribbons 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 that Helly's or Zenker's fixatives were used and that in some cases sections were cut at 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 fine film. 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 San Felice's fluid and stained with crystal violet. RESULTS The chromosomes o/Mayetiola The chromosomes and early cleavage divisions of Mayetiola destructor were investigated by Metcalfe (1935). Although this account is inaccurate in several respects it correctly establishes that chromosome elimination occurs at the fifth division in this species and also that there is no subsequent elimination of chromosomes from the presumptive somatic nuclei in male embryos.