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Surface ultrastucture of the larval hemocytes of the silkworm, Bombyx mori L. (Lepidoptera : Bombycidae)
Int. ]. Insect Morphol. & EmbryoL 5(1): 17-21. 1976. Pergamon Press. Printed in Great Britain.
S U R F A C E U L T R A S T U C T U R E OF THE LARVAL HEMOCYTES OF THE SILKWORM, BOMB YX MOR1 L. (LEPIDOPTERA
• BOMBYCIDAE)
I-/IROMU AKAI a n d SHIGERU SATO The Sericultural Experiment Station, Wada, Sugiuami-ku, Tokyo; and University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo, Japan
(Accepted 16 September 1975) Abstract Hemocytes of Bombyx larvae were studied by scanning electron microscopy using the critical point drying method. The surface structures of the spindle-shaped, leafshaped, and sickle-shaped plasmatocytes are described. Granular cells were covered with numerous cytoplasmic projections. Characteristic details of the surface structure of oenocytoids, spherule cells, and prohemocytes were also studied. The relationship between the surface and internal ultrastructure is indicated. Index descriptors (in addition to those in title) : Critical pointdrying method.
INTRODUCTION INSECT HEMOCYTES are usually g r o u p e d into 5 or 6 m a i n classes: p r o h e m o c y t e s (proleucocytes), plasmatoeytes, g r a n u l a r cells, spherule cells, adipohemocytes, a n d oenocytoids (Jones, 1962; Wigglesworth, 1965): W e classified Bombyx larval hemocytes into 5 classes according to their u l t r a s t r u c t u r e a n d f u n c t i o n (Akai a n d Sato, 1971, 1973) following the earlier classification derived f r o m light microscopic studies (Nittono, 1960). T h e recent use of the critical p o i n t drying m e t h o d for the s c a n n i n g electron microscopic o b s e r v a t i o n has p r o d u c e d excellent, artifact-free specimens o f t h e i n t e r n a l soft tissues o f a n i m a l s ( D u P r a w , 1965; G o l o m b a n d Bahr, 1971). This p a p e r describes the results by s c a n n i n g electron microscopy using critical p o i n t drying method.
MATERIALS AND METHODS The larvae used were hybrids from the cross between 2 varieties of Bombyx mori (J. 1249 × C. 124c~). The silkworms were reared on mulberry leaves in a culture room at 25-27°C. Prior to electron micros topic observation, fresh hemocytes, collected on a glass slide were observed under the light microscope. For observation with the scanningelectron microscope a hind leg of 1-day old 5th instar larva was cut, and drops of hemolymph were collected and fixed for 30 min in cold, buffered 2-5 ~ glutaraldehyde in a centrifuge tube. After fixation, the cells were centrifuged at 500 rpm for 15 min, and then the pellet was washed in several changes of sucrose solution (buffered at pH 7-3). These centrifuged pellets were post-fixed with 1 ~ osmium tetroxide (at pH 7"3) in Veronal buffer for 1 hr. After the fixation, the pellets were dehydrated in graded concentrations of ethanol, and then immersed in amyl acetate solution for 1 hr in order to exchange the ethanol with amyl acetate. The pellets were placed in the specimen chamber of a critical point dryer. Liquid carbon dioxide was poured into the specimen chamber at 25°C, and then the temperature in the chamber was raised above the critical point of carbon dioxide (35-50°C). After the drying, the surfaces of the specimens were coated with carbon and gold in a vacuum evaporator, and photographed in HSM 2 scanning electron microscope. I.M.E.
511--B
17
18
~-IIROMUAKAIand SHIGERLISATO
RESULTS AND DISCUSSION Hemocyte specimens prepared by critical point drying method give a very clear view of their surface structure. In Fig. 1 a small fragment from the pellet is shown at low magnification. Judging from the known internal ultrastructure of Bombyx hemocytes, plasmatocytes, granular cells, and oenocytoids can be distinguished. A typical spindle-shaped plasmatocyte is shown in Fig. 2. Its surface has several small projections, and it corresponds to the Sl plasmatocyte in Fig. 10. Nittono (1960) has pointed out that the plasmatocytes are spindle-shaped for only a few minutes after withdrawal, and they then transform into spherical, oval, pear-shaped, and amoeboid forms. In our specimens, however, transformed plasmatocytes were not detected, because they were fixed so soon after withdrawal of the hemolymph. In Fig. 3 leaf-shaped plasmatocyte is shown. In the transmission electron microscope, it is impossible to observe the entire cell in sections, because it is never in one plane. In Fig. 4 a sickle-shaped plasmatocyte is shown. The cell surface has many wrinkles and a small number of cytoplasmic projections. In our previous paper (Akai and Sato, 1973)we observed the ultrastructure of sectioned spindleshaped plasmatocytes, and subdivided them into thick ($1), medium ($2), and thin (83) forms as shown in Fig. 10. Figure 5 shows adjacent spindle-shaped and leaf-shaped plasmatocytes. The surface of the spindle-shaped cell has undulations, and it may correspond to $2 form shown in Fig. 10. Figure 6 shows granular cells, spindle-shaped plasmatocytes, and spherule cell. The granular cells were covered with numerous cytoplasmic projections, and they are similar to the G I - G 6 stages shown in Fig. 10. Figure 7 shows an oenocytoid, the largest of the Bombyx hemocytes. The cell corresponds to Oi in Fig. 10. Figure 8 shows a prohemocyte or a young plasmatocyte. A typical spherule cell is shown in Fig. 9. Spherule cells (Sp) contain large spherules which appear as bulbous swellings on the cell surface, thus making the spherule cells clearly distinguishable from the other cells in the SEM specimen. It is clear that SEM studies by the critical point drying method support the hemocyte classification arrived at by the ultrastructure of hemocyte sections.
FIGS. 1-9. Scanning electron micr0graphs of larval hemocytes of Bombyx mori, prepared with critical point drying method, FIG. 1. Lower magnification picture of an aggregation of several types of larval hemocytes: plasmatocytes (S), granular cells (G), spherule cells (Sp) and an oenocytoid (O). × 1100 Fro. 2. Typical spindle-shaped plasmatocyte. Several cytoplasmic processes (arrows) are seen on cell surface, x4500 FIo. 3. Leaf-shaped plasmatocyte. Top (right upper corner) and tail (left lower corner) of the cell. x 4500 FIG. 4. Sickle-shaped plasmatocyte, x4500 FIG. 5. Spindle-shaped plasmatocyte lying near a leaf-shaped plasmatocyte, x4500
Larval Hemocytes of the Silkworm
19
20
HIROMU AKAI and SH[GERLI SATO
Fla. 6. Aggregated hemocytes. Granular cells (G) are covered with cytoplasmic projections. × 4500 Fro. 7. Large oenocytoid, x 4500 FIG. 8. Prohemocyte (proleucocyte). x4500 FIG. 9. A typical spherule cell. x 5000
Larval Hemocytes of the Silkworm
131
10
132
21
S~
lo .=
Fro. 10. Schematic drawings of the ultrastructure of the larval hemocytes from ultra-thin sections (Akai and Sato, 1973). Ol : immature oenocytoid containing small amounts of cytoplasmic protein fibers, Oz : mature oenocytoid containing protein fibers deposited in concentric layers, Sp: spherule cell containing large spherules of fine homogeneous material, Gt-G6: granular cells, P1-Pz: prohemocytes, Sl-S3: plasmatocytes which are subdivided into 3 types: thick (Sl), medium ($2), and thin (Sa)spindle-shaped cells. Acknowledgements--We thank Professor Robert C. King, Northwestern University for reading the manuscript. We are also thankful to Mrs. T. Nakayama and K. Matsumoto, Nissei Sangyo Ltd. Co., for assistances with the electron microscopic observations.
REFERENCES AK~a, H. and S. SATO. 1971. An ultrastructural study of the hemopoietic organs of the silkworm, Bombyx mori L. J. Insect Physiol. 17: 1665-77. AICAI, H. and S. SATO. 1973. Ultrastructure of the larval hemocytes of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). Int. J. Insect Morphol. Embryol. 2(3): 207-3•. DUPRAw, E. J. •965. Macromolecular organization Of nuclei and chromosomes. A folded fibre model based on whole-mount electron microscopy. Nature (Lond.) 206: 338-43. GOLOMB,H. M. and G. F. BA~R. 1971. Scanning electron microscopic observations of surface structure of isolated human chromosomes. Science (Wash. D.C.) 171: 1024-26. HORRtD~E, G. A. and S. L. T ~ M . 1969. Critical point drying for scanning electron microscopic study of ciliary motion. Science (Wash. D.C.) 163: 817-18. JONES, C. J. 1962. Current concepts concerning insect hemocytes. Amer. ZooL 2: 209-46. NITTONO, Y. 1960. Studies on the blood cells in the silkworm, Bombyx mori L. Bull. Serieult. Exp. Sta. 16: 171-266. WmOLESWORTH,V. B. •965. The Principles of Insect Physiology, Dutton, New York.
S U R F A C E U L T R A S T U C T U R E OF THE LARVAL HEMOCYTES OF THE SILKWORM, BOMB YX MOR1 L. (LEPIDOPTERA
• BOMBYCIDAE)
I-/IROMU AKAI a n d SHIGERU SATO The Sericultural Experiment Station, Wada, Sugiuami-ku, Tokyo; and University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo, Japan
(Accepted 16 September 1975) Abstract Hemocytes of Bombyx larvae were studied by scanning electron microscopy using the critical point drying method. The surface structures of the spindle-shaped, leafshaped, and sickle-shaped plasmatocytes are described. Granular cells were covered with numerous cytoplasmic projections. Characteristic details of the surface structure of oenocytoids, spherule cells, and prohemocytes were also studied. The relationship between the surface and internal ultrastructure is indicated. Index descriptors (in addition to those in title) : Critical pointdrying method.
INTRODUCTION INSECT HEMOCYTES are usually g r o u p e d into 5 or 6 m a i n classes: p r o h e m o c y t e s (proleucocytes), plasmatoeytes, g r a n u l a r cells, spherule cells, adipohemocytes, a n d oenocytoids (Jones, 1962; Wigglesworth, 1965): W e classified Bombyx larval hemocytes into 5 classes according to their u l t r a s t r u c t u r e a n d f u n c t i o n (Akai a n d Sato, 1971, 1973) following the earlier classification derived f r o m light microscopic studies (Nittono, 1960). T h e recent use of the critical p o i n t drying m e t h o d for the s c a n n i n g electron microscopic o b s e r v a t i o n has p r o d u c e d excellent, artifact-free specimens o f t h e i n t e r n a l soft tissues o f a n i m a l s ( D u P r a w , 1965; G o l o m b a n d Bahr, 1971). This p a p e r describes the results by s c a n n i n g electron microscopy using critical p o i n t drying method.
MATERIALS AND METHODS The larvae used were hybrids from the cross between 2 varieties of Bombyx mori (J. 1249 × C. 124c~). The silkworms were reared on mulberry leaves in a culture room at 25-27°C. Prior to electron micros topic observation, fresh hemocytes, collected on a glass slide were observed under the light microscope. For observation with the scanningelectron microscope a hind leg of 1-day old 5th instar larva was cut, and drops of hemolymph were collected and fixed for 30 min in cold, buffered 2-5 ~ glutaraldehyde in a centrifuge tube. After fixation, the cells were centrifuged at 500 rpm for 15 min, and then the pellet was washed in several changes of sucrose solution (buffered at pH 7-3). These centrifuged pellets were post-fixed with 1 ~ osmium tetroxide (at pH 7"3) in Veronal buffer for 1 hr. After the fixation, the pellets were dehydrated in graded concentrations of ethanol, and then immersed in amyl acetate solution for 1 hr in order to exchange the ethanol with amyl acetate. The pellets were placed in the specimen chamber of a critical point dryer. Liquid carbon dioxide was poured into the specimen chamber at 25°C, and then the temperature in the chamber was raised above the critical point of carbon dioxide (35-50°C). After the drying, the surfaces of the specimens were coated with carbon and gold in a vacuum evaporator, and photographed in HSM 2 scanning electron microscope. I.M.E.
511--B
17
18
~-IIROMUAKAIand SHIGERLISATO
RESULTS AND DISCUSSION Hemocyte specimens prepared by critical point drying method give a very clear view of their surface structure. In Fig. 1 a small fragment from the pellet is shown at low magnification. Judging from the known internal ultrastructure of Bombyx hemocytes, plasmatocytes, granular cells, and oenocytoids can be distinguished. A typical spindle-shaped plasmatocyte is shown in Fig. 2. Its surface has several small projections, and it corresponds to the Sl plasmatocyte in Fig. 10. Nittono (1960) has pointed out that the plasmatocytes are spindle-shaped for only a few minutes after withdrawal, and they then transform into spherical, oval, pear-shaped, and amoeboid forms. In our specimens, however, transformed plasmatocytes were not detected, because they were fixed so soon after withdrawal of the hemolymph. In Fig. 3 leaf-shaped plasmatocyte is shown. In the transmission electron microscope, it is impossible to observe the entire cell in sections, because it is never in one plane. In Fig. 4 a sickle-shaped plasmatocyte is shown. The cell surface has many wrinkles and a small number of cytoplasmic projections. In our previous paper (Akai and Sato, 1973)we observed the ultrastructure of sectioned spindleshaped plasmatocytes, and subdivided them into thick ($1), medium ($2), and thin (83) forms as shown in Fig. 10. Figure 5 shows adjacent spindle-shaped and leaf-shaped plasmatocytes. The surface of the spindle-shaped cell has undulations, and it may correspond to $2 form shown in Fig. 10. Figure 6 shows granular cells, spindle-shaped plasmatocytes, and spherule cell. The granular cells were covered with numerous cytoplasmic projections, and they are similar to the G I - G 6 stages shown in Fig. 10. Figure 7 shows an oenocytoid, the largest of the Bombyx hemocytes. The cell corresponds to Oi in Fig. 10. Figure 8 shows a prohemocyte or a young plasmatocyte. A typical spherule cell is shown in Fig. 9. Spherule cells (Sp) contain large spherules which appear as bulbous swellings on the cell surface, thus making the spherule cells clearly distinguishable from the other cells in the SEM specimen. It is clear that SEM studies by the critical point drying method support the hemocyte classification arrived at by the ultrastructure of hemocyte sections.
FIGS. 1-9. Scanning electron micr0graphs of larval hemocytes of Bombyx mori, prepared with critical point drying method, FIG. 1. Lower magnification picture of an aggregation of several types of larval hemocytes: plasmatocytes (S), granular cells (G), spherule cells (Sp) and an oenocytoid (O). × 1100 Fro. 2. Typical spindle-shaped plasmatocyte. Several cytoplasmic processes (arrows) are seen on cell surface, x4500 FIo. 3. Leaf-shaped plasmatocyte. Top (right upper corner) and tail (left lower corner) of the cell. x 4500 FIG. 4. Sickle-shaped plasmatocyte, x4500 FIG. 5. Spindle-shaped plasmatocyte lying near a leaf-shaped plasmatocyte, x4500
Larval Hemocytes of the Silkworm
19
20
HIROMU AKAI and SH[GERLI SATO
Fla. 6. Aggregated hemocytes. Granular cells (G) are covered with cytoplasmic projections. × 4500 Fro. 7. Large oenocytoid, x 4500 FIG. 8. Prohemocyte (proleucocyte). x4500 FIG. 9. A typical spherule cell. x 5000
Larval Hemocytes of the Silkworm
131
10
132
21
S~
lo .=
Fro. 10. Schematic drawings of the ultrastructure of the larval hemocytes from ultra-thin sections (Akai and Sato, 1973). Ol : immature oenocytoid containing small amounts of cytoplasmic protein fibers, Oz : mature oenocytoid containing protein fibers deposited in concentric layers, Sp: spherule cell containing large spherules of fine homogeneous material, Gt-G6: granular cells, P1-Pz: prohemocytes, Sl-S3: plasmatocytes which are subdivided into 3 types: thick (Sl), medium ($2), and thin (Sa)spindle-shaped cells. Acknowledgements--We thank Professor Robert C. King, Northwestern University for reading the manuscript. We are also thankful to Mrs. T. Nakayama and K. Matsumoto, Nissei Sangyo Ltd. Co., for assistances with the electron microscopic observations.
REFERENCES AK~a, H. and S. SATO. 1971. An ultrastructural study of the hemopoietic organs of the silkworm, Bombyx mori L. J. Insect Physiol. 17: 1665-77. AICAI, H. and S. SATO. 1973. Ultrastructure of the larval hemocytes of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). Int. J. Insect Morphol. Embryol. 2(3): 207-3•. DUPRAw, E. J. •965. Macromolecular organization Of nuclei and chromosomes. A folded fibre model based on whole-mount electron microscopy. Nature (Lond.) 206: 338-43. GOLOMB,H. M. and G. F. BA~R. 1971. Scanning electron microscopic observations of surface structure of isolated human chromosomes. Science (Wash. D.C.) 171: 1024-26. HORRtD~E, G. A. and S. L. T ~ M . 1969. Critical point drying for scanning electron microscopic study of ciliary motion. Science (Wash. D.C.) 163: 817-18. JONES, C. J. 1962. Current concepts concerning insect hemocytes. Amer. ZooL 2: 209-46. NITTONO, Y. 1960. Studies on the blood cells in the silkworm, Bombyx mori L. Bull. Serieult. Exp. Sta. 16: 171-266. WmOLESWORTH,V. B. •965. The Principles of Insect Physiology, Dutton, New York.