Electron microscopy of siliceous spicules from the freshwater sponge Heteromyenia

© 1968 by Academic Press Inc.

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J. ULTRASTRUCXVRERESEARCH22, 12--21 (1968)

Electron Microscopy of Siliceous Spicules from the Freshwater Sponge Heteromyenia RYAN W. DRUM

Department of Botany, University of Massachusetts, Amherst, Massachusetts 01003 Received July 14, 1967, and in revised form November 29, 1967 Siliceous spicules from the freshwater sponge Heteromyenia were studied using three-dimensional carbon replicas. Precise images of spicule structure were obtained. After silica removal with 5 % HF, organic axial threads remained within the spicule replicas. Negative staining of isolated threads reve/tled little or no internal organization. The relationships between the axial threads and spongin, and between polysaccharide surfaces and the biogenesis of siliceous structures are discussed. Sponges deposit silica in distinct structures called spicules. These siliceous parts are generally extracellular and form part of the sponge skeletal framework. They are constructed intracellularly by silica deposition upon an axial thread within a cytoplasmic vesicle (6, 10). We have been primarily concerned with diatoms, unicellular algae which produce a pair of external scales around each cell to form a siliceous exoskeleton (3, 4, 9, 13). Because of their respective similar aquatic growth habits, it seemed possible that sponges might resemble diatoms in the use of silica. Examination of the literature indicated that no previous work had been done on siliceous spicule fine structure. Since most of the taxonomic scheme used to classify freshwater sponges is based on spicule structure, which requires that certain sponges produce only certain spicule forms, an electron microscope study of spicule structure might aid our understanding of taxonomic affinities among sponges as well as the biogenesis of siliceous structures. Techniques used to study diatom silica (4, 9) were modified to prepare carbon replicas of sponge spicules (2), and the results are presented here. MATERIALS AND METHODS Several large (20-50 g) bright green growths of the freshwater sponge Heteromyenia sp. were collected on October 2, 1966, from Hawley Bog near Ashfield, Massachusetts, Spherical Fie. 1. Carbon replica of one-half of a long birotulate; the spicule surface is smooth, without microspines or other irregularities. × 7200.

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yellow gemmules 0.5-1.0 m m in diameter were abundant. F o r spicule studies 20 g of sponge were boiled in concentrated H N O , for 30 minutes, washed several times with distilled water to remove acid, and stored in 30 % aqueous ethyl alcohol. Some were dried on cover glasses and mounted in Hyrax for light microscopical studies. F o r carbon replicas, spicules were shaken into suspension and a drop of this suspension was placed on a Formvar-coated grid (copper or nickel). It was necessary to coat the grids with water-cast Formvar films rather than glass-cast films. Several drops of a 0.2 % ethylene dichloride Formvar solution were dropped onto a clean deionized water surface, the grids placed directly on the resulting film, and then collected with a glass slide; glass-cast Formvar films and carbon films proved unsatisfactory because spicule replicas on them usually collapsed before or during examination in the electron microscope. A 400 ~ layer of carbon was evaporated onto the spicules while they were rotated at least twice through 360 degrees to ensure complete coating (they were placed in a vacuum evaporator on a stage which could be rotated by an external control). The grids were then gently immersed in 5 % H F for 10 minutes, washed several times in deionized water, dried at room temperature, and examined in a Zeiss EM-9 electron microscope. Shadowcasting with palladium-gold did not significantly improve the replica image nor provide further information about spicule structure; it was possible to affect shadowing with regular carbon deposition after formation of the initial replica layer with rotation. To study the internal axial organic component of the spicules, only 50-100 I t carbon was deposited on the spicules during 1-2 rotations through 360 degrees; H F treatment removed the silica and usually most of the delicate replicas; organized intact internal organic threads remained and could be examined directly, negatively stained with 1% PTA, or ashed using the ultramicroincineration technique of Thomas (14) with some modification. Nickel grids with isolated organic threads were coated on the reserve side with silicon monoxide and then placed in a muffle furnace at 650°C for 30 minutes (Fig. 13). The organism was identified from a standard key (8) and from the spicule pictures by Dr. W. Hartman. The author will provide cleaned spicules on request. The unsolicited encouragement of the Sturbridge, Massachusetts Police Department is greatly appreciated.

OBSERVATIONS T w o types of spicules o c c u r r e d in this sponge, two acerate f o r m s (Figs. 2-4) t w o b i r o t u l a t e s f r o m the g e m m u l e s (Figs. 1, 6, a n d 10). A l t h o u g h all f o u r f o r m s illustrated, only the s h o r t b i r o t u l a t e g e m m u l e spicule was studied in detail. W h e n i n t a c t spicules were examined, surface structures were obscured, a n d axial t h r e a d s were i m p o s s i b l e to detect due to c o m p l e t e electron o p a c i t y (Figs. 6

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FIG. 2. Acerate spicule replica showing simple microspines (S) and the single internal vein (V). x 5000. FIG. 3. Simple and compound microspines occur on this spicule; internal veins are associated with many of the spines, x 5000. FIG. 4. Compound microspines (CS) are common on this spicule, but the replica is too thick to permit observation of internal veins, x 5000. FIG. 5. Carbon replica of an opaline phytolith from wheat epidermis. The silica surface is reticulate and much more irregular than that of spicules. × 6300.

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10). Consequently, little or nothing was learned in addition to what was known from light microscope examination. Spicule surface replicas were usually smooth, and the silica did not have any obvious surface patterns. Microspines occurred on both acerates (Figs. 2-4) and on the short birotulate (Figs. 7 and 9), but not on the long birotulate (Fig. 1). Simple microspines occurred on the short birotulate and the more abundant fusiform acerate (Fig. 2); compound and simple microspines occurred on the other acerate (Fig. 4). Inside most spicule replicas were axial threads (V, Figs. 2, 3, 7, and 9). In the simple-spined acerate and the smooth birotulate a single linear thread was usually present. In the compound-spined acerate, linear veins were associated with each compound microspine (Fig. 3). The most complex thread system occurred in the short spiny birotulates; a thick (0.25/~) axial thread passes throughout the length of the spicule shaft as seen in profile (Fig. 7) and connects with m a n y smaller threads (50 m/z) arranged radially 90 degrees from the main axial thread as seen in face view of a rotulate end replica (Fig. 9). There is a separate thread for each rotulate margin ray spine (Figs. 9 and 11). A thin carbon layer attached the spicules to the grid, but did not form a stable replica, and permitted examination of the internal venation separately. The smaller threads were disrupted from flattening in profile (Fig. 8) but retained their orientation in face-on attachment (Fig. 11), and appear to meet at or near the shaft thread end without merging with either each other or the shaft thread. Negatively stained preparations suggest no internal banding pattern (Fig. 12). The thread material was burned away by ultramicroincineration (Fig. 13). The organic debris which remains in a plastic tube after dissolution of the siliceous spicules by 5% H F stained with Congo red, azure B, methylene blue, and crystal violet. Occasionally small disks occurred on short birotulate replicas (Fig. 14) which were not observed on replicas of the other spicule types. We have been unable to obtain thin sections of siliceous spicules; the silica shattered and fell out of the Epon. FIG. 6. Cleaned silicious birotulate spicule which is completely electron opaque (profile view), x 4000. FIG. 7. Three-dimensional profile view carbon replica of a birotulate which has a central vein (V) in the spicule shaft, x 4000. FIG. 8. Isolated internal venation system from a birotulate, x 3800. FIG. 9. Face view of a birotulate end replica which shows a separate radial vein for each marginal ray microspine (ca. 50 each). The shadow effect was caused by carbon deposition at ca. 30 degrees after replica formation, x 5400. FIG. 10. Cleaned birotulate end in face view prior to light carbon coating ((50-100 A) and silica removal (ca. 75 ray spines), x 3000. FIG. 11. Face view of radial birotulate end venation system showing both the radial veins (ca. 75) and the shaft vein. Note the replica fragment at the arrow, x 4500. FIG. 12. PTA negatively stained isolated birotulate veins which show no obvious surface structure or internal regularity. The radial veins are 50 m/z wide and the shaft vein is 250 mE~wide. × 67,000. FIG. 13. Remains of microincinerated birotulate radial veins, x 44,000.

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FIG. 14. Portion of a birotulate end in face view which shows little disks present upon the outer spicule surfaces. × 8000. DISCUSSION The occurrence of axial threads within spicule replicas after silica removal and their isolation on grids substantiates earlier light microscopical observations (6, 10). Thread material staining reactions suggest that it is a carbohydrate, possibly a polysaccharide. (Sufficient quantities of thread material have not yet been obtained for chemical analysis.) Silica is deposited intracellularly upon these threads (6, 10); the same cells which produce siliceous spicules subsequently secrete a fibrous substance, spongin, in which the spicules become emeshed (12). Since the same cells produce both axial threads and spongin, some structural similarity might be expected between the two types of material; spongin resembles collagen, with obvious characteristic transverse banding patterns (5), whereas axial threads as seen here, have no banding patterns. Spongin " A " fibrils are about 40 m/z wide (5), and axial threads to ray spines are about 50 m# wide, this is the major resemblance between them. Bronsted's fibrils (1), are probably spongin (5). Deposition of opaline silica upon polysaccharide surfaces seems to be a crucial factor in the biogenesis of siliceous structures. Most grasses deposit opaline phytoliths (Fig. 5) in leaf and floral inflorescence cells, after these cells reach their maximum size, on the proximal or inner cell wall surface, which is a polysaccharide surface (7). Dead plant material exhibits a similar phenomenon in nature and the laboratory. If dead leaves or twigs (woody stems) fall into or are placed in water with 200-700 ppm of dissolved silicate content, a layer of amorphous opaline silica is deposited

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on all exposed polysaccharide surfaces: when the plant material is incinerated, delicate siliceous replicas of the leaves and twigs remain (15). Petrifaction of wood to form siliceous fossils proceeds in a similar manner. Some evidence exists which suggests that intracellular vesicular silica deposition in diatoms initiates around polysaccharide centers (13). Biogenesis of siliceous structures occurs in either highly evolved modern groups (diatoms and grasses) or in rather ancient, relic groups which appear to be evolutionary culs-de-sac (sponges and Equisetum). Diatoms and grasses are very successful groups, in the water and on land, respectively. Silica is used by all diatoms for cell walls, and by most grasses (and Equisetum) for stem rigidity and flexibility. Not all sponges deposit silica, and only a few extensively; perhaps the role of silica in sponge metabolism, morphology, and survival is critical for certain taxa only, particularly the freshwater sponges. The little disks seen in Fig. 14 may represent a developmental stage or a mistake. No channels were found in the silica as postulated by Schwabe and Wahl (11). Silica deposition to form characteristic spicules is of vital importance to freshwater sponge classification. Although no new characteristics are offered here, the threedimensional replica technique affords a convenient method for obtaining precise spicule images and recording them as permanent photographic records. I recommend that replicas of spicules from all extant freshwater sponge taxa be recorded and future identifications be made with the aid of such a compilation.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

BRONSTED,H. V. and CARLSEN,F. E., Exptl. Cell Res. 2, 90 (1951). DRUM,R. W., Science in press (1967). DRUM, R. W. and PANICRATZ,H. S., ar. Ultrastruct. Res. 10, 217 (1964). DRUM, R. W., PANKRATZ,H. S. and STOERMER,E. F., Electron Microscopy of Diatom Cells, Vol. 6 of Diatomeenschalen im Elektronemikroskopisehen Bild p. 24. Cramer, Lehre, 1966. GRoss, J., SOKAL,Z. and ROUGVl~, M. J., Histochem. Cytochem. 4, 227 (1956). JORGENSEN,C. B., Kgl. Danske Videnskab. Selskab Biol. Medd. 19, (7), 1 (1944). PARRY, D. W. and SMITHSON,F., Ann. Botan. 30, 525 (1966). PENNAK,R. W., Freshwater Invertebrates of the United States. Ronald Press, New York, 1953. REINMANtq,B., LEWlN, J., and VOLCANI,B., or. Phycol. 2, 74 (1966). SCHRODER,K., Z. Morphol. Oekol. Tiere 31, 245 (1936). SCHWABE,G. M. and WAHL, B., Naturwissenschaften 43, 513 (1956). SIMPSON,T. L., J. Exptl. Zool. 154, 135 (1963). STOERMER,E. F., PANKRATZ,H. S. and BOWEN, C., Am. J. Botany 52, 1067 (1965). THOMAS,R. S., Proc. 5th Intern. Congr. Electron Microscopy, Philadelphia, 1962 Vol. 2, RR-11. Academic Press, New York, 1962. VAIn, J. G., Soluble Silicates, Vol. I p. 160. Reinhold, New York, 1951.