A Cytological Study of the Gregarine Parasites of Tenebrio Molitor, Using the Ultra-Centrifuge. by Margaret I

A cytological study of the Gregarine Parasites of Tenebrio molitor, using the Ultra-centrifuge. By Margaret I. Daniels, B.A., M.Sc, Department of Zoology, Trinity College, Dublin. With Plate 28 and 2 Text-figures. INTRODUCTION. THE ultra-centrifuge, developed by J. W. Beams, has been used for much cytological work on metazoan cells and its importance to cytologists can hardly be over-estimated. It provides a new method of attacking problems that formerly relied alone on fixing, staining, and morphological evidence for their solution. As yet, little work has been done using this method on the Protozoa. In America, King and Beams (1934) have investigated the cytoplasm of Paramoecium by this means, and in this department protozoan structure has been examined thus. Patten and Beams (1936) have described the effects of ultra-centrifuging some free-living flagellates, and E. Brown has also worked on Paramoecium (communicated). The present paper deals with Sporozoa, in which a number of different cell inclusions have already been described by various authors. The fact that these particular sporozoans are among those described by Joyet-Lavergne (1926) in a comprehensive paper added further interest to the study. I wish to thank Professor J. Bronte Gatenby for his suggestion of this problem, and for his invaluable advice at all stages of the work. MATERIAL AND TECHNIQUE. The gregarines parasitic in larvae of Tenebrio molitor L. are, in many ways, ideal material for study by this method. The mealworms are cheap, and easy to obtain; they can be kept and fed with little difficulty; they have quite a high percentage 294 MABGARET I. DANIELS of infection by the gregarines; they fit well into the centrifuge rotor, and so can be centrifuged alive. Against all this there is one drawback: it is impossible to tell whether the mealworm is parasitized or not, until the whole process of centrifuging, fixing, embedding, and sectioning has been completed. But, by examining the wax sections under the microscope, it is usually easy to see if parasites are present or not, without staining and mounting the sections permanently. Another difficulty is that of getting good fixation of the parasites, for the fixing fluid has to penetrate the thick gregarine pellicle as well as the cells of the gut-wall of the host. This, however, may be minimized by cutting the gut into very small pieces. The Tenebrio larvae containing the gregarines were sent from London, and conveniently kept in tins with perforated lids. They were fed on crushed oats. Many different fixatives were tried, and it was found that the methods of Champy, Benoit, and Altmann (Baker's modification) gave a satisfactory fixation of the mitochondria. The material may then be stained by the iron alum haematoxylin long method, by the acid fuchsin picric acid method of Altmann, and by the Bensley Cowdry modification of it. The Golgi material is best shown by the osmic acid fixation method of Weigl or Mann-Kopsch. The long fixation method advised by Baker, in Mann's fluid gives good results. After five to six days in osmium tetroxide at 80° C, the material is left in distilled water at the same temperature for 24 to 86 hours. Silver methods, such as those of da Fano, Cajal, and Aoyama, give no satisfactory results in spite of repeated experiments varying the times of immersion in the fixatives, silver nitrate solution, and reducing liquid. The Aoyama cadmium-chloride method, which gave beautiful and consistent results in the cells of the host gut-wall, gave no good results in the gregarines. The fat is preserved by the osmium tetroxide-containing fixatives, and becomes a brown colour. It is also very well shown by the Sudan IV technique after formol saline fixation, recommended by Kay and Whitehead (1985). The paraglycogen is very stable and is beautifully demonstrated by the iodine GREGAKINES OF TENEBBIO 295 gum method advised by Langeron. It provides a very useful way of determining quickly whether parasites are present on a slide or not, for the dark brown colour of the paraglycogen granules is immediately visible. As well as the more usual nuclear stains, Feulgen's nuclear reagent was tried with interesting results. When Champy and Benoit fixed sections were used, the long hydrolysis advised by Painter in the new edition of the 'Microtomist's Vade- Mecum' (1937) was necessary. When centrifuged material was being prepared for cutting wax sections, the three or more centrifuged guts were embedded in one block, while several other uncentrifuged guts were treated in exactly the same way and embedded in another block, as controls. These wax sections were cut 2-5JU. thick. The gregarine material was also studied in fresh smear preparations. Centrifuging Technique. Although the centrifuge top or rotor will actually hold six or seven mealworms, three is the maximum number that should be centrifuged at one time. This allows them enough room to lie in one row along the outer edge of the cavity; if they he two or more deep, the worms nearer the centre will be subject to a lower centrifugal force than the outermost ones. Various forces and times were experimented with, and it was found that at high pressures the gut was often torn out of the worm, breaking at one end (usually the anterior) and remaining attached at the other. The parasites themselves, on account of their thick pellicle, stand the pressure very well. When they have been sufficiently centrifuged—three or four minutes at a pressure of 40 lb. per square inch is usually long enough—the worms are quickly removed from the rotor, the head and last millimetre or two are cut off with scissors and the gut carefully drawn out with forceps. It is best to do this from the hinder end of the animal, so that there is no danger of breaking the anterior region of the mid-gut during the process. It is cut in the region of the Malpighian tubules and the mid-gut part is put, at once, into the fixative, where it is cut into small pieces. 296 MAEGAEBT I. DANIELS The importance of cutting it up into very small parts, or teasing it with a pair of needles, must be stressed, because most of the fixatives containing osmium tetroxide have poor powers of penetration, and it is imperative that the gregarines should be fixed as soon as possible. A generous volume of the fixing fluid should also be used. When experimenting with different centrifugal forces, smear preparations were found very useful, as one can quickly see, without fixing or staining whether sufficient force to move all the paraglycogen granules down to the heavy pole, has been applied or not. R (b) (c) TEXT-FIG. 1. Diagram of the three species found, (a) Gregarina steini; (6) Gregarina cuneata; (c) Gregarina polymorpha. OBSEKVATIONS. Three different gregarine species were found in the gut of the Tenebrio larvae (see Text-fig. 1). At first, I supposed these to be the three species described by Leger and Duboscq in 1904 and Joyet-Lavergne in 1925, namely Steinina ovalis, Gregarina polymorpha Hamm.and Gregarina cuneata Stein, but after reading the relevant literature I believe that Steinina ovalis seldom or never parasitized the mealworms I used, but that Gregarina steini is very common. This question of identification is discussed at greater length below (see p. 315). However, since the cytoplasmic inclusions, and their behaviour when ultra-centrifuged, seem to be similar for the three species, the following account may be taken as general except where it is stated to be specific. Five different kinds of inclusions have been found in the GREGABINES OF TENEBEIO 297 cytoplasm of the gregarines studied, and these have character- istic positions in the eentrifuged animal (see Text-fig. 2). The lightest bodies are the fatty globules; these go to the extreme centripetal pole of a eentrifuged gregarine and share this position with the Golgi material existing in the form of small granules, when this is present. Next come the larger Golgi bodies as irregular platelets and rods. The nucleus takes up a position below the Golgi apparatus and is underlain and TEXT-FIG. 2. Generalized diagram of a eentrifuged gregarine. F., fat; G.A. (a) granular Golgi material; (6) larger Golgi elements; N., nucleus; K., karyosome; M., mitochondria; P., paraglycogen. C, chro- midia. surrounded by the mitochondria. Paraglycogen granules have a greater specific gravity than the mitochondria and go to the centrifugal pole. Chromidial granules may be seen in this region, among the paraglycogen bodies, and have, apparently, a specific gravity very similar to that of the paraglycogen. Fat. Fatty globules are found in all species, but the amount present varies, apparently, ontologically and in different species. The globules themselves may also vary very much in size, and in an uncentrifuged gregarine they are scattered irregularly 298 MARGAKET I. DANIELS throughout the cytoplasm (see figs. 1 and 6, PL 28). They move to the extreme centripetal pole of a centrifuged animal (see figs. 2, 3, and 4, PL 28). They are found both in the deutomerite and in the protomerite and have been seen even in the epimerite (see fig. 1, PL 28). Eegarding the differentiation between the various types of fatty substances, the recent work of Kaufmann and Lehmann as reported by Kay and Whitehead, shows that the usual histological methods, such as Ciaccio, Smith-Dietrich, Fischler, and nile blue are quite unspecific and of little use in accurate work. However, they find two reliable tests that are specific in their action, and can be used to determine fatty substances without having to resort to chemical analysis.

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