GA.METOGENESIS A.NO FK UTILISATION IN NlCMATUS MUESLI. 101 Gametogenesis and Fertilisation in Nematus ribesii. Li. Doncaster, M.A., Late Mackinnon Student of the Royal Society; Lecturer in Zoology in the University of Birmingham. With Plate 8. IN a previous paper1 I gave an account of the maturation and behaviour of the polar nuclei in several species of sawflies which develop parthenogenetically. In all these species there were two maturation divisions, giving rise to an egg nucleus and three polar nuclei, and in some cases fusion took place between the second polar nucleus and the inner half of the first. The egg nucleus sank into the yolk and began to divide to form the embryo, while the polar nuclei in all cases ultimately disintegrated. Since whenever the chromosomes were clearly visible their number appeared to be eight, both in the maturation mitoses and in the later divisions in body-cells, it was concluded that no reduction in the ordinary sense took place. But if fertilisation ever takes place by conjugation of male and female pronuclei, an obvious difficulty arises with regard to the chromosome number in fertilised eggs, and since the process of fertilisation had not been thoroughly examined at the time when the paper referred to was written, it was necessary to leave the question open in the hope of finding a satisfactory answer later. This paper gives an account of 1 'Quart Journ. Micr. Sci.,' vol. 49, 1906, p. 561. 102 L. DONOASTER. the work done on the fertilised egg in Nematus ribesii and on the gametogenesis in that and other species. The methods used were generally the same as before, but it was found that, in searching for male pronuclei in the eggs of impregnated females, thionin or gentian violet were more satisfactory stains than iron hEematoxylin, since they stain nucleus and cytoplasm but leave the yolk uncoloured. In the work on spermatogenesis and the development of the ovarian egg, osmic fixatives (e. g. Flemming's fluid) were largely used in addition to sublimate. THE FERTILISED EGG IN N. RIBESII. In some animals, e.g. the bee, the fertilised egg is easily distinguished from the virgin by the presence of sperm asters in the yolk, but in the sawflies nothing of the kind can be found, and over 200 eggs had to be cut and examined before it became certain that conjugation of male and female pronuclei takes place. In very young eggs I had occasionally found minute rod-like bodies in the peripheral protoplasm near the anterior end, which are probably the heads of spermatozoa, and in somewhat later eggs bodies which appeared to be degenerating nuclei sometimes appear in a similar position. In eggs laid by impregnated females there are frequently in the yolk in front of the polar region more or less numerous small radiating patches of protoplasm which sometimes appear to contain indistinct nuclei, but protoplasmic masses not certainly distinguishable from these are found also in virgin eggs, although with less regularity. In eggs which are probably fertilised there are also fre- quently lines of protoplasm running inward from the edge of the egg near the point where the spermatozoa had been found. But in no case have I been able to recognise with complete certainty the male pronucleus before the maturation divisions of the egg are completed, and after that stage nuclei found in the yolk may always be derived from the OAMETOGENESJS AND FERTILISATION IN NEMATUS RJBESII. 103 egg-nucleus itself. It is never possible, therefore, to say with certainty that a given egg is fertilised or not. But after much time spent in vainly trying to follow the entrance of the spermatozoon aud its conversion into the inale pronucleus, I at last was able to observe the conjugation of the sperm-nucleus with that of the egg, and so to prove that true fertilisation does take place (fig. 1). It occurs immediately after the maturation divisions; the three polar nuclei lie near the edge of the egg (two of them in the same section as the egg and sperm nuclei), and the fusion of the two inner polar nuclei has not yet taken place. The male and female pronuclei are in contact, the male being distinctly smaller than the female, but in another egg in which the same stage is seen the two are of about equal size. The subsequent stages of the conjugation and division of the zygote nucleus have not been observed, but the section represented in fig. 1 leaves no reasonable doubt that normal conjugation takes place. It therefore became necessary to reconsider my previons conclusions with regard to the number of chromosomes, since never more than eight have been found in either fertilised or virgin eggs. I was thus led to work out the spermatogenesis, and to the fresh work on the maturation divisions to be described later. In my previous paper I mentioned that the behaviour of the polar nuclei appeared to be slightly different in fertilised and in virgin eggs, and subsequent work has confirmed this. In the virgin egg of N. ribesii the two inner polar nuclei fuse and give rise to a group of chromosomes, which is generally clearly double, with eight in each half. The two halves of the group do not lie far apart, and commonly remain without much change for some time. But in the majority of eggs from impregnated females the chromosome groups derived from the two inner polar nuclei lie completely and sometimes widely separated, as if the conjugation between the nuclei had been much less complete than in virgin eggs (figs. 2, 3, and 4). Further, in virgin eggs the polar chromosomes usually do not divide, at least for some 104 L. DONOASTEB. time, but in fertilised eggs they frequently divide compara- tively early, giving groups containing as many as sixteen chromosomes rather irregularly arranged in the " polar protoplasm." That this difference in behaviour is really connected with fertilisation is made probable by the fact that it rarely, if ever, occurs in eggs which are certainly virgin, but in the eggs laid by impregnated females it is frequent. Further, in several eggs laid by impregnated females the polar nuclei follow the typical virgin arrange- ment, and in these the little rayed protoplasm masses in the yolk, characteristic of fertilised eggs, are absent; but other eggs laid by the same female have the fertilised type of polar chromosomes, and in these the rayed protoplasm patches are also present. It appears, therefore, that the fertilisation of the egg- nucleus, or the presence of spermatozoa in the egg, in some way influences the behaviour of the polar nuclei. SPERMATOGENESIS. When it had been shown that normal fertilization could take place in N. ribesii, it became necessary to re-examine the maturation divisions in order to make certain about the chromosome number, which I asserted in the previous paper to be eight both in the maturation and in the somatic mitoses, and also apparently in fertilized eggs. The matura- tion of the egg begins immediately after it is laid, so that it is very difficult to get good preparations of the early stages, and I therefore decided to examine the matter first iu the development of the spermatozoa. In very young male pupae, shortly after the larval skin is cast in the cocoon, the testes consist of compact groups of cells at the sides of the alimentary canal. These cells (spermatogonia) have relatively large nuclei containing a conspicuous nucleolus (plasmosome) and eight or about eight chromatin masses apparently attached to the nuclear mem- brane (fig. 5). Division figures are scarce, but when found GAMET0GENES1S AND FERTILISATION IN NEMATUS BIBESH. 105 they show clearly about eight rather large chromosomes in the equatorial plate, which split so that eight travel towards each centrosome (fig. 6 a, b). At a later stage the testis becomes larger, and consists of lobes or compartments in each of which all the cells are in about the same stage. By the time the colours of the mature fly are beginning to appear the testis contains nothing bnt spermatids and nearly mature spermatozoa, but when the pupa is still white all stages from spermatogonia to spermatids are found in dif- ferent lobes, often in the same section. In the nucleus before the first maturation divisions the chromatin consists of a number of irregular masses (appa- rently about eight, but they are always rather indistinct). Shortly afterwards it becomes condensed into four more concentrated masses, each of which frequently appears double or quadruple (fig. 7 a,b, c). A spindle is then formed, and the four chromatic masses become tightly packed together in the equatorial plate, which is much smaller than in the sperniatogonial divisions. There are conspicuous centro- somes. The chromosomes in the spindle are so tightly packed together that it is difficult to be certain of their number, but a comparison of many mitoses leaves little doubt that there are four, each of which is bivalent (6g. 8 a, b). The mitosis appears to be of the heterotype form, resembliug the figures found by Moore in the cockroach 1 except that the chromo- somes are fewer and very much smaller (fig. 9 ft, b). They are, however, appreciably larger than the chromosomes in the maturation mitoses of the egg. The second maturation division is easily distinguished from the first by the fact that the spindle is of about half the diameter; the chromosomes are usually even more tightly packed, so as frequently to appear as a siDgle body, but in clearer cases there is little doubt that there are four (fig.