Ultrastructure of Rumen Holotrichs by Electron Microscopy M. D. STERN, W. H. H O O V E R , and J. B. L E O N A R D 1 Department of Animal and Veterinary Sciences University of Maine Orono 04473

ABSTRACT

ovoid form and large size with dense, longitudinal rows of cilia. The two species differ principally in the position of the cytostome, but their overall structure is almost identical. In lsotricha intestinalis, the cytostome is in the postero-lateral portion of the body (6) while the cytostome of Isotricha prostoma is located posteriorly but not laterally. The one species of Dasytricha, ruminantium (76/2 × 44/2), is smaller than the isotrichs and commonly occurs in greater numbers in the tureen (4). Holotrichs are less complex ciliates than entodiniomorphs, and study of the ultrastructure of holotrichs by transmission electron microscopy (TEM) has been minimal. Noirot-Timoth~e (7) studied the ectoplasmendoplasm boundary of Isotricha using light microscopy and TEM. She indicated that the cytoplasm is divided into two zones, ectoplasm and endoplasm, which are separated by a discontinuous double layer consisting of large fibrillar strands. The lack of information pertaining to the ultrastructure of holotrichs leaves this area open to exploration. The current study was to elucidate some ultrastructural characteristics of holotrichs by TEM.

Thin sections of rumen ciliated protozoa of the subclass Holotrichia were observed with a transmission electron microscope. These protozoa had a double layered boundary separating the ectoplasm from the endoplasm. Starch granules were abundant throughout the endoplasm and are probably storage starch. Also in the endoplasm of the holotrichs were the macro- and micronuclei. They were adjacent to one another, apparently surrounded by a continuous membrane. Many unidentified dense bodies appeared in the endoplasm adjacent to the inner layer of the ectoplasmic-endoplasmic boundary. These inclusions could be precursor material to the boundary. The cuticle contained granular inclusions which might be secreted to facilitate ciliary movement. The holotrichs ingested chloroplasts as these were in vacuoles throughout the cytoplasm. The anal pore appeared to be open to the exterior and lined by a unit membrane. INTRODUCTION

Ciliated protozoa of the subclass Holotrichia (holotrichs) found most frequently in the rumen of sheep and cattle are those of the genera lsotricba and Dasytricba. Imai and Tsunoda (6) used the scanning electron microscope to observe the surface structures of ciliated protozoa in sheep rumen. They observed that the two species of Isotricha, prostoma (151/2 × 95/2) and intestinalis (173/2 × 127/2), are ciliates of

Received November 3, 1976. t Department of Anatomical Sciences, State University of New York, Buffalo, NY 14214. 2Cellulose source, from Nutritional Biochemical Corp., Cleveland, OH. 911

MATERIALS AND METHODS

Rumen contents were obtained from a fistulated sheep 2.5 h after being fed a semi-purified pelleted diet. A fistulated sheep was used because in a previous study of a fistulated steer, hol0trichs were n o t present (8). The percentage composition of the diet as fed was: sucrose, 2.7; corn starch, 23.9; Alphacel 2 , 28.8; peanut meal, 20.0 (IRN 5-03-650); ground hay, 20.0 (IRN 1-02-274); urea, 1.4; dicalcium phosphate, 1.0; and animal fat, 2.2. Rumen protozoa (holotrichs) were prepared for TEM by the procedure of Stern et al. (8). Thin sections were observed in a Philips EM-201 at an accelerating voltage of 60 kv.

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FIG. 1A. A transverse section of Dasytricba ruminantium showing the starch granules (s), food vacuole (f), cilia (c), and the double layered b o u n d a r y (bd) between the endoplasm and ectoplasm. T h e inset reveals bacteria in vesicles as indicated b y the arrows. × 3150; inset, × 8 6 5 0 . B. A transverse section of Isotricba intestinalis showing the esophagus (e), mierorubules (mr) of the cytostome, macronucleus (rna), and contractile vacuole (cv). × 3900. Journal of Dairy Science VoL 60, No. 6

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FIG. 2A. Electron micrograph of an tsotricba which has accumulated an abundance of starch. The cytoplasm consists of food vacuole (f) containing numerous bacteria and a macronucleus (ma) with invaginations (i). × 3000. B. Electron micrograph of a section through the endoplasm of Dasytricba rurninantium. The micronucleus (mi) is adjacent to the macronucleus (ma) and appears to be surrounded by a continuous membrane (m). The darker staining nuclear material, compared to Fig. 2A, is probably an artifact of preparation. × 11,900. Journal of Dairy Science Vol. 60, No. 6

c~

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91 5

FIG. 3A. Electron micrograph of a section through the ectoplasmic-endoplasmic boundary region of an

lsotricba. The ectoplasm (ec) is separated from the endoplasm (en) by a double layer (bd). The dense bodies (d) are possibly precursor material for the double layer. × 10,500. B. Electron micrograph of the cuticular region of a holotrich. An extension of the cuticle (cu) occurs between each cilium (c) and within the cuticles are unidentified granular inclusions (u). Heavy arrows indicate granular inclusions apparently in the process of being exuded from the cuticles. × 28,000. C. A cross section of the esophagus (e) of Dasytricba ruminantium. × 17,000.

RESULTS A N D DISCUSSION

Strained rumen fluid was examined with a light microscope prior to fixation. Holotrichs and entodiniomorphs both were present; however, the holotrichs appeared to be more prevalent. Figure 1A is an electron micrograph of a transverse section of Dasytricha rurninantium and shows the shape and general structure of the organism. Cilia surround the whole body surface area. The ectoplasm is separated from the endoplasm by a double layered boundary. While discontinuity is in the double layer, as suggested by Noirot-Timothee (7), the double layer does not appear to consist of fibrillar strands. Vacuoles are throughout the organism and may be either contractile, as determined by the opaque appearance, or food vacuoles with

chloroplasts, bacteria, or food particles. Polysaccharide grains are abundant throughout the cytoplasm and are probably storage starch. Similar to Dasytricha in gross structure, the Isotricba intestinalis also is ciliated densely and has a discontinuous double layer separating the ectoplasm and endoplasm (Fig. 1B). Isotricba intestinalis is characterized mainly by its large size and the location of the cytostome in a postero-lateral position. The cytostome, however, did not appear to be equidistant between the posterior end of the cell and the middle as suggested by Hungate (4). The holotrichs use soluble sugars and convert them rapidly into stored polysaccharide which is used during periods when sugars are not available (2). Hungate (5) has suggested Journal of Dairy Science Vol. 60, No. 6

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FIG. 4. Electron rnicrograph of a holotrich vacuole containing chloroplasts (ch). Digestion of the chloroplasts in this micrograph appears minimal as most of the grana stacks (gs) and lamellae (1) remain intact. X21,000.

that holotrichs have n o t evolved a mechanism to control the storage of starch. This theory is supported by observations of Sugden and Oxford (9), who found that when holotrichs are provided with excess sugar, they store starch until they burst. Figure 2A shows an lsotricba which is populated densely with storage starch. In addition to the copious amount of starch granules in the cytoplasm, the food vacuole is populated profusely with bacteria. The macronucleus is similar to that of other tureen protozoa except for the continuous outer membrane which appears as invaginations into the macronucleus. These invaginations have not been reported before, and their function has not been determined. In Dasytricba rurninantium the mieronucleus and macronucleus are adjacent to one another a n d appear to be encapsulated i n a continuous membrane structure (Fig. 2B). in contrast with the entodiniomorphs (8), the nuclei of holotrichs are located Journal of Dairy Science Vol. 60, No. 6

in the endoplasm and n o t in the ectoplasm. The ectoplasmic-endoplasmic boundary appears distinctly in Fig. 3A. The boundary thickness varies from .6 to .7/1, which agrees with Noirot-Timothee (7), who found a thickness varying from .4 to .7/~ according to the angle of the section. Many unidentified dense bodies appear in the endoplasm adjacent to the inner layer of the ectoplasmic-endoplasmic boundary. These inclusions could be a precursor material to the boundary. While this is only speculative, the consistent localization of these inclusions on the inside of the endoplasmic border supports this theory. An extension of the cuticle occurs between each cilium on the body surface (Fig. 3B). Imia and Tsunoda (6) found that the cilia measured .2/a in width and 5.8/.t in length. In our study the cilia also measured .2/a in width, but length measurements were not determined. The cuticle was .3~ in width and .8/a in length.

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FIG. 5. Electron micrograph of an anal pore (ap) of an lsotricha. The pore is open to the exterior and is lined by a unit membrane (u). × 15,000.

The cuticle contains inclusions which appear granular in substance. The proximity of these granular structures to the outer edge of the cuticles suggests that they are secreted to the outside of the cuticle. Instances can be observed in Fig. 3B where the inclusions appear to be migrating toward the top of the cuticle or are penetrating through the side of the cuticle. The secretion might lubricate the ciliary region facilitating movement of the cilia. Each cuticle contains at least two of these inclusions. These inclusions are n o t exclusively in cuticles which surround the body surface as they also line the esophageal wall (Fig. 3C). The esophagus is lined with cilia, which suggests that the inclusions are located in areas which specifically contain cilia. This is supported by Stern et al. (8), who found that the cuticular region of entodiniomorphs where cilia were n o t present did not contain these inclusions.

Chloroplast digestion has been studied in entodiniomorphs (3, 10) but has not been observed in holotrichs. West and Mangan (10) reported that chloroplasts were ingested rapidly by entodiniomorphs, and although ammonia was not produced, the formation of 8-aminovaleric acid indicated protein degradation. The ability of holotrichs to ingest chloroplasts is evident in Fig. 4 which shows a vacuole containing numerous chloroplasts. Many of the chloroplasts are intact with the grana stacks of closely packed lamellae clearly visible. The soluble material of the chloroplasts probably is released in the vacuole, degraded, and assimilated by the protozoan. Therefore, chloroplast amino acids probably are incorporated into protozoal protein as are bacterial amino acids (1). Undigested particles are eliminated from the celt through the anal pore. At the posterior end of the cell, the anal pore is visible as a distinct Journal of Dairy Science Vol. 60, No. 6

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inpocketing of the cuticle (Fig. 5). The pore is a p p r o x i m a t e l y 6.3~ in length and 1.4/2 in width as measured at the opening of the anal pore. The pore itself is c o m p l e t e l y open to the exterior and appears to be lined by a single unit membrane. This study revealed that although holotrichs are n o t considered c o m p l e x ciliates, they do possess intriguing ultrastructural characteristics. These p r o t o z o a had a double layered b o u n d a r y separating the ectoplasm f r o m the endoptasm. Vacuoles were t h r o u g h o u t the organism and contained either chloroplasts, bacteria, or f o o d particles. Starch granules were a b u n d a n t t h r o u g h o u t the endoplasrn. The heterogeneous nature of the f o o d s in these organisms is indicative of the holotrich's capacity to ingest a wide variety of material in addition to starch. Many unidentified dense bodies appeared in the endoplasm adjacent to the inner layer of the ectoplasmic-endoplasmic boundary. These inclusions were theorized to be precursor material to the double layer. The cuticle c o n t a i n e d granular inclusions which appeared to be secreted possibly to facilitate ciliary m o v e m e n t . These inclusions were n o t exclusive to the cuticular area, as they also lined the esophageal wall. The use of T E M in this study has enabled us to observe various ultrastructural features of the holotrichs which had not been observed previously by light microscopy.

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REFERENCES

1 Coleman, G. S. 1972. The metabolism of the amino acids of Escberichia coli and other bacteria by the tureen ciliate Entodinium caudatum, J. Gen. Microbiol. 47:449. 2 Forsyth, G., and E. L. Hirst. 1953. Protozoal polysaccharides. Structure of the polysaccharide produced by the holotrich ciliates present in sheep's rumen. J. Chem. Soc. 2132. 3 Hall, F. J., J. West, and G. S. Coleman. 1974. Fine structural studies on the digestion of chloroplasts in the rumen ciliate Entodinium caudatum. Tissue & Cell 6:243. 4 Hungate, R. E. 1966. The Rumen and Its Microbes. Academic Press, New York. 5 Hungate, R. E. 1975. The rumen microbial ecosystem. Ann. Rev. of Ecol. and Systematics 6:39. 6 Imai, S., and K. Tsunoda. 1972. Scanninge|ectron microscopic observations on the surface structure of ciliated protozoa in sheep rumen. Nat. Inst. Anim. Hlth. Quart. 12:74. 7 Noirot-Timothee, C. 1958. L'ultrastructure de la limite ectoplasme-endoplasme et des fibres formant le caryophore chez les cilies du genre lsotricha stein. Compt. Rend. 247:692. 8 Stern, M. D., W. H. Hoover, R. G. Summers, Jr., and J. H. Rittenburg. 1977. Ultrastructure of ~rumen entodiniomorphs by electron microscopy. J. Dairy Sci. 60:902. 9.Sugden, B., and A. E. Oxford. 1952. Some cultural studies with holotrich ciliate protozoa of the sheep's rumen. J. Gem Microbiol. 7:145. 10 West, J., and J. L. Mangan. 1972. The digestion of chloroplasts in the rumen of sheep and the effect of disruption and gtutaraldehyde treawaent. Proc. Nutr. Soc. 31:108A.

Ultrastructure of rumen holotrichs by electron microscopy.

Ultrastructure of Rumen Holotrichs by Electron Microscopy M. D. STERN, W. H. H O O V E R , and J. B. L E O N A R D 1 Department of Animal and Veterina...
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