Ultrastructure of Rumen Entodiniomorphs By Electron Microscopy M. D. STERN, W. H. H O O V E R , R. G. SUMMERS, Jr) and J. H. R I T T E N B U R G Department of Animal and Veterinary Sciences University of Maine Orono 04473
ABSTRACT
neath the cuticle consists of an outer membrane which covers a homogeneous second layer and an innermost layer which is composed of transversely oriented filaments. Terminology of the tubular-appearing elements referred to as filaments has not been consistent. These tubular-appearing elements have been described as "fiber," (9) "fibril," (10) "filament," (13) and "microtubule" (4). We have chosen the term microtubule as suggested by Coleman and Hall (4). These microtubules located beneath the cuticle may be capable of sliding past one another and may serve as a transport mechanism or may influence cuticle contractions. Retraction of the adoral cilia accompanied by retraction of the retrociliary microtubules (9) frequently interferes with gross ultrastrucrural studies in this area. This retraction is caused by exposure to unfavorable conditions (6) such as those encountered in preparation for microscopy studies. While the major objective of this study was description of the ultrastructure of entodiniomorphs as revealed by transmission electron microscopy (TEM), a technique to prevent retraction of the adoral cilia was evaluated also.
Thin sections of rumen ciliated protozoa of the subclass Spirotrichia were studied by electron microscopy to elucidate their ultrastructure. To prevent retraction of their adoral cilia, menthol crystals were used to relax the retrociliary region. These protozoa had a distinct ectoplasm and endoplasm with the macroand micronuclei located in the ectoplasm. At the surface of the entodiniomorph b o d y was a highly differentiated cortical zone of four layers. Ribosomes were abundant throughout the cytoplasm, suggesting a substantial potential for protein synthesis. These protozoa appeared to engulf bacteria into l a n e vacuoles, and subsequently the bacteria were taken into the endoplasm in vesicles containing only one bacterium each. The bacteria were digested partially, and only in isolated cases were the bacterial cell walls still intact. INTRODUCTION
The ultrastructure of the protozoa species in the tureen has n o t been investigated extensively. Rumen protozoa of the subclass Spirotrichia (entodiniomorphs) are complex ciliates, and further definition of their structure may enhance understanding how protozoa function. In addition, observations of the nutrients ingested and degraded will provide insight into the role of protozoa in rumen metabolism. Several species of entodiniomorphs have been studied by electron microscopy (1, 2, 10, 11). These studies revealed that the region be-
M A T E R I A L S A N D METHODS
Received November 3,1976. 1Department of Anatomical Sciences, State University of New York, Buffalo, NY 14214.
Rumen contents were from a fistulated steer 1 h after feeding an 80% timothy hay-20% ground corn diet. Rumen contents were strained slowly through four layers of cheesecloth which had been treated with 5 to 6 g of menthol crystals by sprinkling on the surface of the cloth. Protozoa were exposed to these menthol crystals as a means of relaxing the retrociliary region and preventing contraction of the adoral cilia. A separatory funnel containing 1 liter of this strained tureen fluid was placed in an incubator at 39 C for 45 min. After removal from the incubator the protozoal sludge which had settled to the b o t t o m of the flask was removed and centrifuged for 5 rain at 100 × g.
902
ULTRASTRUCTURE OF ENTODINIOMORPHS
903
FIG. 1. A transverse section of Entodinium caudaturn showing the esophagus (e), macronucleus (ma), food vacuole (f), starch granules (s), bacteria in vesicles(b), and the boundary between the endoplasm and ectoplasm (bd). X4200. Protozoa in the resultant pellet were fixed at 10 C for 1 h in 3% glutaraldehyde dissolved in .1 M phosphate buffer pH 6.5. The cells then were post-fixed in 2% osmium tetroxide for 1 h. The pellet was washed for 1 h in three changes of phosphate buffer. D e h y d r a t i o n consisted of o n e 10-min change of 70%, two 10-rain changes of 95%, and three 30-rain changes of absolute ethanol followed by two 30-rain changes of propylene oxide. The protozoa were embedded in Araldite 506, and the resin was hardened at 60 C for 3 days. Sections were cut with a Cambridge-Huxley ultramicrotome, mounted on copper grids, and stained with lead citrate (12). The sections were observed in an RCA EMU-3G electron microscope at an accelerating voltage of 50 kv or in a Philips EM-201 at an accelerating voltage of 60 kv.
RESULTS A N D DISCUSSION
Examination of the ciliated protozoa by light microscopy prior to fixation revealed that protozoa of the subclass Holotrichia were not present. The protozoa species most frequently observed were E n t o d i n i u r n c a u d a t u m and E n t o d i n i u m bursa. Figure 1 is an electron micrograph of a transverse section of E n t o d i n i u m c a u d a t u m and shows the general structure of the organism. The ectoplasm contains the nucleus, and the endoplasm contains bacteria in vesicles, which have been ingested by the protozoan. The ectoplasm and endoplasm appear to be divided by a discontinuous boundary. The boundary does not appear to be a unit membrane as suggested by Coleman and Hall (3). The esophagus, which contains cilia, is separated from the endoplasm by a clearly defined membrane. The ectoplasm contains, in
FIG. 2A. Electron micrograph "of the cortical zone of an entodiniomorph consisting of four layers. These layers are the outer membrane (o), homogeneous layer (h), microtubule layer (mt), and the inner membrane (i). The cytoplasm consists of endoplasmic reticulum (er), starch granules (s), glycogen (g), and the granular appearance is due to the abundance of ribosomes. × 25,000. B. Electron micrograph of the microtubules (mr) at oblique angles to the cuticle. Unidentified cytoplasmic granular inclusions (gi) were also observed. × 27,000.
Journal of Dairy Science Vol. 60, No. 6
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ULTRASTRUCTURE OF ENTODINIOMORPHS
905
FIG. 3A. Electron micrograph of a section through the adoral cilia (c) of an entodiniomorph. Note the osmiophilic structures (w) at the base of the cilia attached to the basal bodies (bb). Retrociliary~microtubules (r) extend from the base of the cilia into the cytoplasm. ×9000. B. Electron micrograph of the cytoplasmic region beneath the base of the esophagus. × 14,000. Journal of Dairy Science Vol. 60, No. 6
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ABSTRACT
neath the cuticle consists of an outer membrane which covers a homogeneous second layer and an innermost layer which is composed of transversely oriented filaments. Terminology of the tubular-appearing elements referred to as filaments has not been consistent. These tubular-appearing elements have been described as "fiber," (9) "fibril," (10) "filament," (13) and "microtubule" (4). We have chosen the term microtubule as suggested by Coleman and Hall (4). These microtubules located beneath the cuticle may be capable of sliding past one another and may serve as a transport mechanism or may influence cuticle contractions. Retraction of the adoral cilia accompanied by retraction of the retrociliary microtubules (9) frequently interferes with gross ultrastrucrural studies in this area. This retraction is caused by exposure to unfavorable conditions (6) such as those encountered in preparation for microscopy studies. While the major objective of this study was description of the ultrastructure of entodiniomorphs as revealed by transmission electron microscopy (TEM), a technique to prevent retraction of the adoral cilia was evaluated also.
Thin sections of rumen ciliated protozoa of the subclass Spirotrichia were studied by electron microscopy to elucidate their ultrastructure. To prevent retraction of their adoral cilia, menthol crystals were used to relax the retrociliary region. These protozoa had a distinct ectoplasm and endoplasm with the macroand micronuclei located in the ectoplasm. At the surface of the entodiniomorph b o d y was a highly differentiated cortical zone of four layers. Ribosomes were abundant throughout the cytoplasm, suggesting a substantial potential for protein synthesis. These protozoa appeared to engulf bacteria into l a n e vacuoles, and subsequently the bacteria were taken into the endoplasm in vesicles containing only one bacterium each. The bacteria were digested partially, and only in isolated cases were the bacterial cell walls still intact. INTRODUCTION
The ultrastructure of the protozoa species in the tureen has n o t been investigated extensively. Rumen protozoa of the subclass Spirotrichia (entodiniomorphs) are complex ciliates, and further definition of their structure may enhance understanding how protozoa function. In addition, observations of the nutrients ingested and degraded will provide insight into the role of protozoa in rumen metabolism. Several species of entodiniomorphs have been studied by electron microscopy (1, 2, 10, 11). These studies revealed that the region be-
M A T E R I A L S A N D METHODS
Received November 3,1976. 1Department of Anatomical Sciences, State University of New York, Buffalo, NY 14214.
Rumen contents were from a fistulated steer 1 h after feeding an 80% timothy hay-20% ground corn diet. Rumen contents were strained slowly through four layers of cheesecloth which had been treated with 5 to 6 g of menthol crystals by sprinkling on the surface of the cloth. Protozoa were exposed to these menthol crystals as a means of relaxing the retrociliary region and preventing contraction of the adoral cilia. A separatory funnel containing 1 liter of this strained tureen fluid was placed in an incubator at 39 C for 45 min. After removal from the incubator the protozoal sludge which had settled to the b o t t o m of the flask was removed and centrifuged for 5 rain at 100 × g.
902
ULTRASTRUCTURE OF ENTODINIOMORPHS
903
FIG. 1. A transverse section of Entodinium caudaturn showing the esophagus (e), macronucleus (ma), food vacuole (f), starch granules (s), bacteria in vesicles(b), and the boundary between the endoplasm and ectoplasm (bd). X4200. Protozoa in the resultant pellet were fixed at 10 C for 1 h in 3% glutaraldehyde dissolved in .1 M phosphate buffer pH 6.5. The cells then were post-fixed in 2% osmium tetroxide for 1 h. The pellet was washed for 1 h in three changes of phosphate buffer. D e h y d r a t i o n consisted of o n e 10-min change of 70%, two 10-rain changes of 95%, and three 30-rain changes of absolute ethanol followed by two 30-rain changes of propylene oxide. The protozoa were embedded in Araldite 506, and the resin was hardened at 60 C for 3 days. Sections were cut with a Cambridge-Huxley ultramicrotome, mounted on copper grids, and stained with lead citrate (12). The sections were observed in an RCA EMU-3G electron microscope at an accelerating voltage of 50 kv or in a Philips EM-201 at an accelerating voltage of 60 kv.
RESULTS A N D DISCUSSION
Examination of the ciliated protozoa by light microscopy prior to fixation revealed that protozoa of the subclass Holotrichia were not present. The protozoa species most frequently observed were E n t o d i n i u r n c a u d a t u m and E n t o d i n i u m bursa. Figure 1 is an electron micrograph of a transverse section of E n t o d i n i u m c a u d a t u m and shows the general structure of the organism. The ectoplasm contains the nucleus, and the endoplasm contains bacteria in vesicles, which have been ingested by the protozoan. The ectoplasm and endoplasm appear to be divided by a discontinuous boundary. The boundary does not appear to be a unit membrane as suggested by Coleman and Hall (3). The esophagus, which contains cilia, is separated from the endoplasm by a clearly defined membrane. The ectoplasm contains, in
FIG. 2A. Electron micrograph "of the cortical zone of an entodiniomorph consisting of four layers. These layers are the outer membrane (o), homogeneous layer (h), microtubule layer (mt), and the inner membrane (i). The cytoplasm consists of endoplasmic reticulum (er), starch granules (s), glycogen (g), and the granular appearance is due to the abundance of ribosomes. × 25,000. B. Electron micrograph of the microtubules (mr) at oblique angles to the cuticle. Unidentified cytoplasmic granular inclusions (gi) were also observed. × 27,000.
Journal of Dairy Science Vol. 60, No. 6
o
o
,-]
X~ Z
c~ ,-]
ULTRASTRUCTURE OF ENTODINIOMORPHS
905
FIG. 3A. Electron micrograph of a section through the adoral cilia (c) of an entodiniomorph. Note the osmiophilic structures (w) at the base of the cilia attached to the basal bodies (bb). Retrociliary~microtubules (r) extend from the base of the cilia into the cytoplasm. ×9000. B. Electron micrograph of the cytoplasmic region beneath the base of the esophagus. × 14,000. Journal of Dairy Science Vol. 60, No. 6
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.
