polyhedroses of the alfalfa caterpillar and other insects. Grote, and the alfalfa caterpillar, Colias philodice eurytheme Bdvl., were used.

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1 A DEMONSTRATION OF THE NATURE OF POLYHEDRA USING ALKALINE SOLUTIONS' KENNETH M. HUGHES University of California, Berkeley, California Received for publication November 1, 1949 The composition of the polyhedral bodies formed within the tissues of insects infected by certain viruses has been the subject of many studies. Glaser and Chapman (1916) believed that polyhedral bodies were composed of an inner dense substance and a lighter peripheral substance. Dikasova (1942) concluded that the polyhedron was an aggregation of ovoid particles but did not state whether or not she believed these to be the virus particles. She also considered this aggregation of particles to be confined within a "lipoid-protein membrane." Bergold (1947) refers to findings of Brill and Kratky to the effect that 82 per cent of the weight of the polyhedron is noninfectious protein and another 5 per cent is the virus. In a communication to Steinhaus (1949), Bergold has stated that about 95 per cent of the polyhedron consists of a noninfectious nucleoprotein and that the remaining 5 per cent is infectious virus. The virus particles of some of the polyhedral viruses have been shown to be rod-shaped particles that may occur in bundles of several rods per bundle (Bergold, 1947; Steinhaus, 1949). That polyhedra will dissolve in a weak alkali has been well established. Glaser and Chapman noted this characteristic in Bergold purified the virus of the silkworm and certain other polyhedroses by dissolving the polyhedral bodies in dilute sodium carbonate. Similar methods have been used successfully in the Laboratory of Insect Pathology, University of California, in studying the polyhedroses of the alfalfa caterpillar and other insects. MATERIALS AND METHODS The polyhedra of two insects, the yellow-striped armyworm, Prodenia praefica Grote, and the alfalfa caterpillar, Colias philodice eurytheme Bdvl., were used. To prepare material for electron microscope study, a diseased larva was triturated in distilled water in a Waring blender. The suspension thus produced was filtered through gauze to remove the larger pieces of tissue debris and was then washed in distilled water by centrifuging for a few minutes in a clinical centrifuge. After two or three such washings, the suspension of polyhedral bodies was usually found to be fairly free of debris. Droplets of this suspension were then placed on the film of electron microscope mounts prepared in the usual manner and were allowed to dry. When they were thoroughly dry, a 0.01 M sodium hydroxide solution was placed on the film and allowed to stand for varying lengths of time. The sodium hydroxide was then drawn off, and the mounts were washed immediately by being touched to the surface of distilled water. When dry, the mounts were 1 A contribution from the Laboratory of Insect Pathology, Division of Biological Control, College of Agriculture, University of California, Berkeley, California. 189

2 1KENNETH M. HUGHES 190 [VOL. 59 ready for examination with the electron microscope. In most cases specimens were shadowed with gold before examination. Other suspensions of washed polyhedral bodies were studied with the dark-field microscope by flowing the sodium hydroxide solution under the cover glass and watching the changes that took place in the polyhedra. RESULTS Dissolution of the polyhedral bodies of the two insects studied shows that these bodies are made up of at least three components: (1) a thin sheath of gelatmous-appearing substance that, under the action of the base, may swell but does not dissolve in the concentrations of sodium hydroxide used; (2) an inner substance that constitutes the major bulk of the polyhedron and that dissolves in the sodium hydroxide; and (3) the virus particles arranged in bundles of several rods each that lie within the inner substance and that are released by the dissolution of the latter. Usually the outer envelope remains intact while the inner substance goes into solution. When this is the case, and when dissolution is complete, all that remains of the polyhedron is an envelope containing a number of bundles of virus particles (figure 1C). When the dissolving process is not complete, an envelope containing pieces of undissolved polyhedral material may be seen (figure 2H). Virus bundles are often visible scattered among these undissolved fragments. Sometimes the envelope appears to rupture and swell away from the surface of the inner substance leaving the latter behind as one undissolved piece or as a number of partly dissolved pieces. This effect is seen more frequently with the electron microscope than with the dark-field microscope, indicating that it may result from stresses during the final drying of the specimen. With the dark-field microscope it is possible to observe most of the details described above. With this instrument, untreated polyhedra appear as brightly shining, crystallike bodies (figure 1A). Upon exposure to sodium hydroxide they lose their uniform luster and become a globule of bright, rapidly moving granules (figure 1B). The outer envelope is only barely visible with dark-field equipment. The virus bundles within are easily discernible and appear violently agitated by Brownian movement. The envelope often remains intact for many minutes before it finally breaks and liberates the dancing virus bundles. A difference in appearance between the virus bundles found in Prodenia and those found in Colias is noticeable with the dark field. The former appear to reflect much more light than do the latter under such conditions, so that the virus bundles of Prodenia appear as brightly shining granules whereas those of Colias are much more difficult to distinguish. Polyhedra do not always exhibit the dissolving phenomenon just described. Sometimes some of the polyhedra dissolve while others do not. In some preparations the only effect of the alkali is to distort the shape of the bodies, resulting in projections extending from the surface of the body (figure 2C). Sometimes the bodies merely fragment into a few large pieces. At times the bodies remain

3 1950] NATURE OF POLYHEDRA 191 Figure 1. A. Dark-field micrograph of Prodenia polyhedra untreated. B. Dark-field micrograph of Prodenia polyhedra after treatment with sodium hydroxide. C to G. Electron micrographs of Prodenia polyhedra after treatment with sodium hydroxide. Gold-shadowed. The outer envelope and the enclosed virus bundles are visible. E and G show envelopes from which all or most of the virus bundles have escaped. F shows virus bundles that have begun to separate into individual particles. Magnifications: A and B, 930 X; C, D, E, and G, 7,050 X; F, 11,600 X.

4 192 KENNETH AM. HUGHES [VOL. 59 Figuire 2. A. Fragments of Prodenia polyhedra, only slightly dissolved after sodium hydroxide treatment, showing cavities or light areas in the inner substance. B. Fragments of Colias polyhedra showing the same phenomenon as A. C. Polyhedron of Prodenia showing projectionis caused by treatment with sodium hydroxide. D. Partially dlissolved polvhedron of Prodenia. The inner substance has broken into pieces. The envelope is not evident. E. Polyhedron of Colias after complete dlissolution of the inner substance with sodium hydroxide. Two virus bundles remain in the envelope. F, G, and H. Polyhedra of Colias after sodium hydroxide treatment. Incomplete dissolution of inner substance has left pieces of this substance remaining within the envelopes. All, except B, shadowed with gold.l Magnifications: A, 10,300 X; B, C, E, F, G, and H, 11,000 X; D, 11,600 X.

5 1950] NATURE OF POLYHEDRA 193 completely unaltered after long exposure to the sodium hydroxide. There are at least two variable factors involved in these differing results. One of these is the age of the original suspension of polyhedral bodies. If the polyhedra have been dried and resuspended, or if they have been held in a distilled water suspension for only a few hours, they do not dissolve readily, if at all. In one case a sample of polyhedra examined with the dark-field microscope dissolved readily within about two or three minutes after the sodium hydroxide solution was introduced under the cover slip. Three hours later another sample of the same preparation was examined. During the intervening time the polyhedra had been left in suspension in distilled water. The polyhedra of the second smple failed to dissolve after being exposed to the same concentration of sodium hydroxide for more than a half-hour. Another factor influencing the rate of dissolution of the polyhedral bodies is the age of the sodium hydroxide solution. Although the solutions used were made up in distilled water and stored in glass flasks, an older solution would not produce the same results as a fresh solution. A fresh solution would dissolve fresh polyhedral bodies in 1 to 2 minutes; a solution several weeks old would require 10 to 20 minutes; and a solution several months old was found to have no visible effects on polyhedra in exposures of an hour. In general, a fresh sodium hydroxide solution placed on fresh polyhedral bodies results in the dissolving phenomenon. Use of an older sodium hydroxide solution or an older polyhedral suspension may result in some fragmentation, some distortion of shape, or no visible change whatever. There is a distinct difference between the two species studied in the relative number of virus bundles in each polyhedron. Electron micrographs of polyhedra of Colias have never shown more than three or four bundles within an envelope. Whether these envelopes are intact or ruptured usually cannot be determined with certainty. With dark-field equipment, Colias polyhedra appear to contain perhaps eight or ten bundles. On the other hand, large polyhedra from Prodenia can be seen to contain over a hundred virus bundles (figure 1D). There appears to be a correlation between the size of polyhedra and the number of virus bundles contained. This is noticeable in the two species studied, for the Prodenia polyhedra are much larger, on the average, than are Colias polyhedra and contain more virus bundles than the latter. The same seems to apply to polyhedra of varying sizes within a species, for large Prodenia polyhedra appear to contain more bundles than do small Prodenia polyhedra. The number of individual virus particles per bundle has not been satisfactorily determined. Partly separated bundles of the Prodenia virus seem to show six members, although this number may not be constant. DISCUSSION The contention of Glaser and Chapman (1916) and Dikasova (1942) that polyhedral bodies are composed of an inner substance surrounded by an outer envelope seems to be upheld. It is to be noted, however, that this effect was apparent in the present work only when fresh polyhedra and fresh sodium hydroxide were used. The sheath apparently hardens rapidly with exposure to air or to pure

6 194 KENNETH M. HUGHES [VOL. 59 water so that it may break with the fragmentation of the inner substance, but it does not swell away from the latter so as to become visible as a separate entity. That the outer covering is a definite structure rather than merely the surface of the polyhedron seems to be evident in photographs (figures 1E and IG). Further evidence is shown by the fact that the outer envelope remains after the inner substance has dissolved; that it forms a limiting wall that contains the virus bundles; that the latter, agitated by Brownian movement, are seen to impinge against the inner surface of the envelope without penetrating it; and that, finally, with a break in the envelope, the virus bundles are liberated rapidly into the surrounding medium. Bergold (1947) published a photograph of a piece of polyhedral body of Porthetria dispar after treatment with sodium carbonate. In the interior of the body can be seen openings or less dense areas that are approximately the size and shape of virus bundles. The same phenomenon has been observed in the polyhedra of both insects used in the present study (figures 2A and 2B). This effect is apparently seen only with older polyhedral bodies that fragment with little or no actual dissolution of the inner substance. Dikasova's (1942) observation that the interior of the polyhedron is made up of separate ovoid pieces seems to have its counterpart in the present study in polyhedra that are only incompletely dissolved. In such cases the dissolution of the inner substance does not take place uniformly but rather acts to break this substance down into a number of separate pieces that may be roughly ovoid in shape (figure 2D). These pieces of the inner substance, however, are not to be confused with the virus particles that have been shown to occur in bundles and that present a quite different appearance from that of the undissolved pieces of polyhedra. Several workers, most notably Paillot (1924, 1926), have reported seeing bright, dancing granules in tissues and hemolymph from diseased insects and in filtrates of suspensions of polyhedral bodies. The appearance of the bundles of virus particles as seen with the dark-field microscope in the present work seems to agree with descriptions by earlier workers sufficiently well to aume that in all probability they were seeing virus bundles. Hence, Paillot's belief that the bright granules he described in the silkworm were the causative virus seems to be upheld, and the name (Borrelina bombycis) he proposed for that virus to be a valid one. A discussion of the granules reported by various men is presented by Steinhaus (1949). SUMBMRY Polyhedral bodies of two insects were exposed to dilute sodium hydroxide for varying lengths of time and were examined with the electron microscope and dark-field microscope. These studies showed that polyhedral bodies are apparently made up of three components: (1) an outer envelope, (2) a dense inner substance, and (3) bundles of virus particles contained in the inner substance. The alkaline solution ordinarily dissolves only the inner substance leaving the envelope enclosing many virus bundles. With the dark-field microscope the virus

7 1950] NATURE OF POLYHEDRA 195 bundles appear to be identical with the bright granules reported by Paillot and others. REFERENCES BEIRGOI, G Die Isolierung des Polyeder-Virus und die Natur der Polyeder. Z. Naturforsch., 2b, DIAsovA, E. T The nature of the polyhedra of the jaundice of the silkworm. Bull. biol. mdd. exptl. U.R.S.S., 13, 101. (In Russian.) GLAsER, R. W., AND CAPMAN, J. W The nature of the polyhedral bodies found in insects. Biol. Bull., 30, PALLOT, A Sur l'dtiologie et l'dpiddmiologie de la grasserie du ver A soie. Compt. rend., 179, 229. PAILLOT, A Existence de la grasserie chez les papillons de ver A soie. Compt. rend. acad. agr. France, 12, STEINHAUS, E. A Principles of insect pathology. McGraw-Hill Book Co., New York.