THE EFFECT OF THE GENE ABDOMEN ROTATUM ON THE DEVELOPMENT OF DROSOPHILA MELANOGASTER NORMAN P. MARENGO AND RUTH B. HOWLAND WashingtonSquare College of Arts and Science New York University Received May 22, I942 INTRODUCTION ECENTLY considerable emphasis in genetic research has been cen- R tered on the effects of genes upon development. The determination of the time at which a gene begins to act and of the manner in which it expresses its specific action in terms of cell or tissue structure has led to a better understanding of the mechanism of gene activity. This is especially true of carefully controlled mutant stocks of Drosophila melanogaster, about which investigations have already yielded much important informa- tion. A number of gene mutations in Drosophila melanogaster causing ab- dominal rotation have been reported in the literature from time to time, but no record of the embryological picture of their effects has appeared. BRIDGESand MORGAN(1923) described a mutant showing abdominal rotation which was in all cases to the left, through 60 to 90 degrees. This mutation was determined to be a simple recessive and was located in chromosome 111. Aside from the counter-clockwise rotation of the ab- domen in the adult, no structural changes were observed. This stock has been lost, due both to poor viability and to difficulty in mating. In 1928 DEMEREC(1938) found a mutant, twisted, located on chromosome I. The abdomen was twisted through 30 degrees in a manner similar to the first described mutant. In 1932 MOHR (1938) discovered a similar mutation located on the same chromosome as DEMEREC’Stwisted. The second case showed more extreme twisting than the first. A third somewhat similar mutant was also described by MOHR,but it is questionable if it was any different from the second “twisted” described. BELIAJEFF(1931). found and named the mutation abdomen rotatum which was used in this study. He determined it to be a recessive located on chromosome IV. The abdominal rotation is clockwise, generally through 45 to 60 degrees (fig. 4). The mutant was first observed during an investiga- tion of wild populations at the INSTITUTEFOR EXPERIMENTAL BIOLOGY at Moscow. BELIAJEFFstated that the twisting was first observed as the development of the flies neared the end, the pupal abdomen taking on an abnormal position within the puparium. A free space was found to appear in the right side of the puparium, the pupal abdomen being pressed against GENETICSa?: 604 Nov. 1942 ABDOMEN ROTATUM IN DROSOPHILA 605 the left wall. No asymmetry or abnormality of the larvae or puparia was seen. BRIDGES(1935) described still another mutant showing abdominal rotation, later determined to be an allele of the abdomen rotatum of BELIAJEFF.The rotation was similar in direction to BELIAJEFF’S,but re- versed its direction as the adult flies aged. With the exception of this brief account by BELIAJEFF,investigators have described the effects of these genes in terms of external adult structures only. For this reason, the present investigation was undertaken to de- termine the effects of the gene abdomen rotatum on stages earlier than the adult. Special attention was also given to the position and condition of the internal organs in histological preparations. MATERIALS AND METHODS The stock used was ar/eyD,a balanced lethal of abdomen rotatum with eyeless dominant. It was secured through the courtesy of DR. M. DEMEREC of the CARNEGIEINSTITUTION OF WASHINGTON, Cold Spring Harbor, N. Y. Each generation of this stock theoretically should have one fly out of three with the rotation. This proportion was cut down considerably by the apparently poor viability of the developing mutants. Several attempts were made to breed a pure w/ar stock, but all except one failed. The few offspring obtained in this instance were not sufficient for use in the study. The rotated females are fertile, but matings involving the males‘ are usually sterile, presumably because of the distortion of the male copulatory organs (BELIAJEFF1931). Crosses of rotated females with known hetero- zygous males produced rotated cases in approximately fifty percent of the offpsring. Repeated crosses of this type furnished most of the material studied. The Oregon R stock from the WASHINGTONSQUARE COLLEGE laboratories provided control material for comparison and contrast. Living material was examined under a binocular dissecting microscope, both with direct light on a black background and with transmitted light. The cultures were kept at a temperature of 25 f 2’ centigrade. Individuals being observed during metamorphosis were kept on microscope slides in moist chambers during the observation periods. Numerous photographs were taken as checks upon recorded observations. For histological study, fixation in Carnoy’s strong fluid or Carnoy- Lebrun was employed, with subsequent treatment according to the usual paraffin method. Sections were cut from six to ten micra in thickness and stained with Delafield’s or Heidenhain’s hematoxylin. In sectioning pupae, it was found that removal of the puparium by dissection prior to infiltra- tion resulted in better sections. Some total mounts of larvae, prepupae, Dissections of adult males in a solution of one-third sea water to two-thirds distilled water (HOWLAND1932) showed the vasa efferentiafilled with actively moving sperm. 606 NORMAN P. MARENGO AND RUTH B. HOWLAND and pupae were made. Staining by both the Feulgen reaction and borax- carmine was employed. These total mounts, however, showed little that could not be seen by direct observation of living material. For study of puparium markings, fragments of puparia were washed in alcohol, cleared in xylene, and mounted in gum damar. The prepupal stage referred to frequently in this paper includes the I 14 hour period between puparium formation and the termination of pupation movements (ROBERTSON1936). .OBSERVATIONS Larvae Observations of larvae confirm BELIAJEFF’Searlier findings as to the lack of any asymmetry at this stage. Study of larvae was limited to living material from ar/eyD stock matings and backcrosses. As yet no method has been found for separating normal larvae from larvae destined to be- come rotated adults. Prior to the time of puparium formation, normal and genetically rotated individuals are indistinguishable. EXPLANATIONOF PLATE FIGUREI.-Genetically “rotated” prepupa with gas bubble (bu). Note persistent segmentation (P.s.) of puparium. FIGURE2.-Rotated pupa. Note asymmetrical position of pupal abdomen within symmetrical puparium. FIGURE3 .-Empty puparium of a rotated individual. Note segmentation of puparium (P.s.) and marked posterior constriction (P.c.). FIGURE4.-Rotated adult male. FIGURE[;.-Normal prepupa prior t; formation of gas bubble. Note smooth contour of pu- parium as contrasted with that of (I). FIGURE6.-Normal pupa. FIGURE7.-Empty puparium of normal individual. FIGURE&-Normal adult male. FIGURE9.-Cross section of normal prepupa through genital disc (g) region. FIGUREIo.-Cross section through genital disc (g) region of genetically “rotated” prepupa. Note symmetrical character of lateral bulges (b) in puparium. Genital disc (8) and tracheae (t) show no rotation. FIGUREI 1.-Cross section of genetically “rotated” prepupa through abdominal region, an- terior to genital disc. FIGURE12.-Cross section through testis region of normal male pupa. FIGURE13. Cross section through testis region of rotated pupa. Note rotation of testes (te) and imaginal discs (i) of abdominal hypodermis. FIGURErq.-Drawing of a section of the dorsal surface of a normal puparium. Markings shown run transversely. FIGUREI[;.--Similar drawing of the puparium of a rotated fly. ABBREVIATIONSUSED b, abnormal bulge in lateral wall of puparium. bu, prepupal gas bubble. h, abdominal hypo- dermis. i, imaginal disc of abdominal hypodermis. 1, leg. m, lateral dorso-ventral muscle. p, pu- parium. p.~.,abnormal posterior constriction of puparium. p.~.,abnormal persistent segmentation of puparium. pp, prepupal cuticle. sp, abnormal space between rotated pupal abdomen and right wall of puparium. t, trachea. te, testis. PLATE ABDOMEN ROTATUM IN DROSOPHILA 607 Particular attention was given to the position of the genital disc in third instar larvae. ROBERTSON(1936) states that at the time of puparium formation this disc is the only imaginal disc in the abdominal region. It lies in the midline of the ventral surface, just anterior to the anus, and is readily seen in living tbird instar larvae. No deviation of this disc was ob- served in the material examined, although the stock was known to con- tain both normal and “rotated” individuals. Puparia The termination of the third larval instar in the normal fly is character- ized by a shortening of the larva and an increase in its diameter as the larval cuticle is transformed into a puparium (ROBERTSON1936). At this stage appears the first character on the basis of which an accurate separa- tion of normal from rotated forms is possible. In normal individuals, the segmental constrictions of the larvae are lost, and the purparium contour becomes quite smooth (fig. 5 and 7). In individuals known to be genetically rotated, marked segmental constrictions remain after the puparium has completely hardened (fig. I and 3, P.s.). These constrictions are con- sistently present and are identified readily as soon as complete quiescence of the larva has occurred. A second puparium abnormality is a deeply marked constriction near the posterior end just anterior to the spiracles (fig. 3, P.c.). There is a slight