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The life cycle of Nosema bombycis as revealed in tissue culture cells of Bombyx mori
IOURXAL
OF INVEBTEBBATE
The Life
PATHOLOGY,
14, 316-320
(1969)
Cycle of Nosem bombycis as Revealed Culture Cells of Bombyx mori’ REN Insect Sault
ISHIHARA~
Pathology Ste. Marie, Received
in Tissue
Research Ontario,
March
Institute, Canada
31. 1969
Nosema bombycis spores inoculated into cultures of Bombyx mori cells resulted in infection of the cells. The development of the pathogen through its complete cycle to the formation of new spores was observed. A form having two nuclei, similar to those of the sporoplasm, was seen among the newly formed spores. This form leaves the host cell, migrates to a new cell, which it penetrates and infects, and the growth cycle is believed to be responsible for spreadis repeated. This “secondary infective form” ing the infection within the host.
ated by Kramer (1960) and Lom and Vavra ( 1963), found that the germ is extruded from its spore through the polar filament. He considered that the germ is injected directly into the host cell by means of the polar filament. If this is the case, the first step of intracellular development is the initself, and the jection of the germ “planont” is an imaginary entity. Trager (1937) successfully infected tissue-culture cells with N. bonrbycis, using blood from diseased insects as inoculum. Although he assumed that the “infective form” in the blood was the “planont,” he did not present the morphologic features of his “infective form” and the blood used as inoculum could have contained hemocytes infected with N. bombycis. An attempt was made to fill the gaps of knowledge on the development of N. bombycis, using silkworm tissue cultures inoculated with spores of this protozoan.
Although the main features of the life cycle of the microsporidian Nosema boml~ycis (the cause of pebrine in the silkworm, Bombz~x nzori) have been known for more than half a century, knowledge of some details, e.g. “planont” stage, is still incomplete. Stempell (1909) coined the term “planont” to refer to the stage between the “germ” (or sporoplasm) discharged from its spore and the intracellular stage (“meront”). He claimed that this stage of the organism multiplies in either the alimentary canal or the hemocoel of the host insect before it penetrates into various tissues of the host to start intracellular development. However, Omori (1912) and Kudo (1916) failed to find the “planont,” and Ohshima (1937) observed that the germ, when extruded into the gut juice of host insect, disintegrates quickly. Ohshima, substanti1 Contribution No. 124 from the Insect Pathology Research Institute, Canada Department of Fisheries and Forestry, Sault Ste. Marie, Ontario, Canada. 3 Present address: Department of Agriculture and Veterinary Sciences, Nihon University, Setagaya-ku, Tokyo, Japan.
MATERIALS
AND
METEIODS
The materials and methods have been described in a previous paper (Ishihara and Sohi, 1966). 316
LIFE CYCLE OF Nosema bombycis OBSERVATIONS
The cultured cell, fixed and stained with Giemsa’s solution just after inoculation of Nosema bombycis spores, harbors binucleate organisms (Fig. 1). The appearance of this stage is similar to that of the germ found outside the cultured cells. The two nuclei are round and compact, and are stained deep red by Giemsa’s solution while the cytoplasm takes up the dye so faintly that the parasite can be recognized only by the presence of the nuclei. During the first several hours after inoculation, the protozoan increases in size keeping its original round appearance. Its cytoplasm stains deep blue in contrast to the case found just after inoculation. As the organism grows, the nuclei gradually become less compact and more irregular in their outline (Figs. 2 and 3). Any “germ” remaining outside host cells either shrinks, leaving two nuclei with a small amount of cytoplasm around them, or becomes swollen with a loose mass of threadlike material in the nuclei. Multiplication of the germ was not observed outside the host cells. The protozoan, after it grows to a certain point, becomes elongate (Figs. 4 and 5). Before this stage, replication did not occur. The elongate form increases in number by bimary fission. Schizogony is rare, if any. Precocious division of nuclei without cytoplasmic separation may be the cause of formation of quadrinucleate forms or chains of binucleate ones (Figs. 5 and 6). Cytoplasm of the host cell is the main site of infection, but its nucleus is also infected with the protozoan. It is quite difficult to detect the organism before it reaches the elongate stage, when observed under a phase microscope. After that, the host cell infected with numerous elongate forms and spores is easily found (Fig. 9). The steps of typical spore formation are shown in Fig. 7. Another type of spore formation, though rare, in which a sporont
317
gives rise to two spores, is shown in Fig. 8. Time of spore formation is different from culture to culture. It may be controlled by the condition of the cultures. Spores tend to be formed earlier at the periphery and the extended part of the host cell. These observations suggest that cellular condition triggers the sporogony. Generally sporogony was recognized 2 to 4 days after inoculation. A form possessingtwo nuclei, round and compact similar to those of the germ, was recognized among the organisms giving rise to spores (Fig. 10). This form is found to leave the host cell (Fig. ll), start migrating (Fig. 12), reach and penetrate a new host cell (Fig. 13), and commence its growth there (Fig. 14). I believe this form is responsible for spreading the protozoa from one cell to the other within the host. It seems to appear in the host cell where the protozoan starts forming spores. Before sporogony starts, this form (which may be called the “secondary infective form”) does not appear. It differs from the gemi, which was either discharged into the culture medium or newly injected itself into the host cell, in its bigger size, variable shape and stronger affinity for the dye. DISCUSSION
Although Stempell (1909) claimed that he found the “planont” in the hemocoel of the host insect two days after inoculating the insect with N. bombycis, it is possible he identified the “secondary infective form” as the “planont,” because of its morphologic similarity. Iwabuchi’s ( 1913) observation that the “planont” penetrates the epithelium of the ovariole of Bombyx mori can be interpreted in the same way. Trager (1937) was not able to infect tissue culture with N. bombycis when the inoculum was taken from the insect 1 day after inoculation. He found that the “infective form” exists in the blood from the insect 2 days after inoculation (but did not say how long this condi-
318
ISHIHARA
LIFE
CYCLE
OF
Nosema bombzJcis
tion persisted ) . His inoculum possibly contained the form which I described above, though his brief report does not state whether his inoculum was free from hemocytes carrying the protozoan. Present experiments confirmed that the “secondary infective form” does not appear in the host cell 1 day after inoculation of N. bombycis. Although I believe the form I found is responsible for spreading the protozoa from cell to cell, I do not know how it can leave the host cell, migrate, and invade the other host cell. The study of fine structure of both the protozoan and host cell may reveal the answer to these questions. This report adds another example that a protozoan which has been classified in the genus Nosema can produce two spores from one sporont. Foa ( 1924), Paillot ( 1928, 1939), and Steinhaus and Hughes ( 1949) have already reported such cases with other Microsporidia as well as with N. bombycis. The frequency of this type of spore formation should be studied in various species of Microsporidia under controlled conditions. Tissue culture technique provides a suitable method for such studies, __Development of all figures
319
ACICNOWLEDGMENT
I express my thanks to Dr. S. S. Sohi of the Insect Pathology Research Institute, who provided the primary cell cultures used in these experiments. REFERENCES
FOA, A.
1924. Quoted from Paillot (1928). ISHIHARA, R., AND SOHI, S. S. 1966. Infection of ovarian tissue culture of Bombyx mori by Nosema bombycis spores. J. Invertebrate Pathol., 8, 538-540. IWABUCHI, H. 1913. Histological studies on the transovarian infection of Nosema bombycb. Tokyo Sangyo-Koshujo Hokoku (Bull. SangyoKoshujo Tokyo) No. 48, l-65. J. P. 1960. Observations on the KRAMER, emergence of the microsporidian sporoplasm. J. Insect Pathol., 2, 433-439. Kuw, R. R. 1916. Contribution to the study of parasitic protozoa. I. On the structure and of Nosema bombycis Naegeli. life history Bull. Imper. Seric. Expt. Stat. Tokyo, 1, 31-51. Lo&r, J., AND VAVHA, J. 1963. The mode of sporoplasm extrusion in microsporidian spores. Acta Protozool., 1, 81-90. OHSHIMA, K. 1937. On the function of the polar filament of Nosema bombycis. Parasitology, 29, 220-224. oxroar, J. 1912. Zur Kenntnis des Pebrine-Er-
__of Nosema bombycis in tissue culture is 1100X, except Fig. 9 which is 350X.
cells from
ovaries
of Bom.byx
mori.
Magnification
FIG. 1. N. bombycis just after invasion (arrow). Only two nuclei are apparent. FIG. 2. Developing N. bombycti (arrow), with cytoplasm colored deeply blue by Giemsa’s solution. FIG. 3. More advanced stage (arrow) with increased size and original round shape. Nuclei somewhat less compact and irregular in shape. FIG. 4. N. bombycis developed to the elongate form without fission. FIG. 5. Fission of N. bombycis (arrow) after development to the elongate form without fission, FIG. FIG.
6. Elongate from actively dividing in host ccl1 cytoplasm. 7. Steps of typical spore formation. Letters a to f indicate the steps in order of development. FIG. 8. Exceptional method of spore formation where two spores are formed from a sporont. FIG. 9. A host cell infected with spores (sp) and elongate form (nb) of the protozoan as observed under an inverted-phase microscope; N: nucleus of the host cell. FIG. 10. Appearance of infective form (IS) in a host cell. FIG. 11. Infective form (IS) leaving a host cell.
FIG. 12. FIG. 13. FIG. 14.
Infective Infective Infective
form form form
(IS) (IS) (IS)
migrating penetrating developing
from host cell. a new host cell. in a host cell after
invasion.
320
ISHIHARA
Nosema bombycis Naegeli. Arb. Kais. Gesundh., 40, 108-122. PAILLOT, A. 1928. Sur le cycle kvolutif de Nosema bombycis, parasite de la p&brine du Ver B soie. Compt. Rend. Sot. Biol., 99, 81-83. PAILLOT, A. 1939. Le carpocapse dans le region lyonnaise et les regions limitrophes. Ann. Epiphyties Phytogen., 5, 199-211. STEINHAUS, E. A., ASD ~ICJGHES, I(. M. 1949. ragers,
Two newly described species of Microsporidia from the potato tuberworm, Gnorimoschema operculella ( Zeller ) ( Lepidoptera, Gelechiidae). J. Yarasitol., 35, 67-74. STEMPELL, W. 1909. Ueber Nosema bombycis Naegeli. Arch. Protistenk, 16, 281-358. THAGER, W. 1937. The hatching of spores of Nosemu bombycis Naegeli and the partial development of the organism in tissue cultures. J. Parasitol., 23, 226-227.
OF INVEBTEBBATE
The Life
PATHOLOGY,
14, 316-320
(1969)
Cycle of Nosem bombycis as Revealed Culture Cells of Bombyx mori’ REN Insect Sault
ISHIHARA~
Pathology Ste. Marie, Received
in Tissue
Research Ontario,
March
Institute, Canada
31. 1969
Nosema bombycis spores inoculated into cultures of Bombyx mori cells resulted in infection of the cells. The development of the pathogen through its complete cycle to the formation of new spores was observed. A form having two nuclei, similar to those of the sporoplasm, was seen among the newly formed spores. This form leaves the host cell, migrates to a new cell, which it penetrates and infects, and the growth cycle is believed to be responsible for spreadis repeated. This “secondary infective form” ing the infection within the host.
ated by Kramer (1960) and Lom and Vavra ( 1963), found that the germ is extruded from its spore through the polar filament. He considered that the germ is injected directly into the host cell by means of the polar filament. If this is the case, the first step of intracellular development is the initself, and the jection of the germ “planont” is an imaginary entity. Trager (1937) successfully infected tissue-culture cells with N. bonrbycis, using blood from diseased insects as inoculum. Although he assumed that the “infective form” in the blood was the “planont,” he did not present the morphologic features of his “infective form” and the blood used as inoculum could have contained hemocytes infected with N. bombycis. An attempt was made to fill the gaps of knowledge on the development of N. bombycis, using silkworm tissue cultures inoculated with spores of this protozoan.
Although the main features of the life cycle of the microsporidian Nosema boml~ycis (the cause of pebrine in the silkworm, Bombz~x nzori) have been known for more than half a century, knowledge of some details, e.g. “planont” stage, is still incomplete. Stempell (1909) coined the term “planont” to refer to the stage between the “germ” (or sporoplasm) discharged from its spore and the intracellular stage (“meront”). He claimed that this stage of the organism multiplies in either the alimentary canal or the hemocoel of the host insect before it penetrates into various tissues of the host to start intracellular development. However, Omori (1912) and Kudo (1916) failed to find the “planont,” and Ohshima (1937) observed that the germ, when extruded into the gut juice of host insect, disintegrates quickly. Ohshima, substanti1 Contribution No. 124 from the Insect Pathology Research Institute, Canada Department of Fisheries and Forestry, Sault Ste. Marie, Ontario, Canada. 3 Present address: Department of Agriculture and Veterinary Sciences, Nihon University, Setagaya-ku, Tokyo, Japan.
MATERIALS
AND
METEIODS
The materials and methods have been described in a previous paper (Ishihara and Sohi, 1966). 316
LIFE CYCLE OF Nosema bombycis OBSERVATIONS
The cultured cell, fixed and stained with Giemsa’s solution just after inoculation of Nosema bombycis spores, harbors binucleate organisms (Fig. 1). The appearance of this stage is similar to that of the germ found outside the cultured cells. The two nuclei are round and compact, and are stained deep red by Giemsa’s solution while the cytoplasm takes up the dye so faintly that the parasite can be recognized only by the presence of the nuclei. During the first several hours after inoculation, the protozoan increases in size keeping its original round appearance. Its cytoplasm stains deep blue in contrast to the case found just after inoculation. As the organism grows, the nuclei gradually become less compact and more irregular in their outline (Figs. 2 and 3). Any “germ” remaining outside host cells either shrinks, leaving two nuclei with a small amount of cytoplasm around them, or becomes swollen with a loose mass of threadlike material in the nuclei. Multiplication of the germ was not observed outside the host cells. The protozoan, after it grows to a certain point, becomes elongate (Figs. 4 and 5). Before this stage, replication did not occur. The elongate form increases in number by bimary fission. Schizogony is rare, if any. Precocious division of nuclei without cytoplasmic separation may be the cause of formation of quadrinucleate forms or chains of binucleate ones (Figs. 5 and 6). Cytoplasm of the host cell is the main site of infection, but its nucleus is also infected with the protozoan. It is quite difficult to detect the organism before it reaches the elongate stage, when observed under a phase microscope. After that, the host cell infected with numerous elongate forms and spores is easily found (Fig. 9). The steps of typical spore formation are shown in Fig. 7. Another type of spore formation, though rare, in which a sporont
317
gives rise to two spores, is shown in Fig. 8. Time of spore formation is different from culture to culture. It may be controlled by the condition of the cultures. Spores tend to be formed earlier at the periphery and the extended part of the host cell. These observations suggest that cellular condition triggers the sporogony. Generally sporogony was recognized 2 to 4 days after inoculation. A form possessingtwo nuclei, round and compact similar to those of the germ, was recognized among the organisms giving rise to spores (Fig. 10). This form is found to leave the host cell (Fig. ll), start migrating (Fig. 12), reach and penetrate a new host cell (Fig. 13), and commence its growth there (Fig. 14). I believe this form is responsible for spreading the protozoa from one cell to the other within the host. It seems to appear in the host cell where the protozoan starts forming spores. Before sporogony starts, this form (which may be called the “secondary infective form”) does not appear. It differs from the gemi, which was either discharged into the culture medium or newly injected itself into the host cell, in its bigger size, variable shape and stronger affinity for the dye. DISCUSSION
Although Stempell (1909) claimed that he found the “planont” in the hemocoel of the host insect two days after inoculating the insect with N. bombycis, it is possible he identified the “secondary infective form” as the “planont,” because of its morphologic similarity. Iwabuchi’s ( 1913) observation that the “planont” penetrates the epithelium of the ovariole of Bombyx mori can be interpreted in the same way. Trager (1937) was not able to infect tissue culture with N. bombycis when the inoculum was taken from the insect 1 day after inoculation. He found that the “infective form” exists in the blood from the insect 2 days after inoculation (but did not say how long this condi-
318
ISHIHARA
LIFE
CYCLE
OF
Nosema bombzJcis
tion persisted ) . His inoculum possibly contained the form which I described above, though his brief report does not state whether his inoculum was free from hemocytes carrying the protozoan. Present experiments confirmed that the “secondary infective form” does not appear in the host cell 1 day after inoculation of N. bombycis. Although I believe the form I found is responsible for spreading the protozoa from cell to cell, I do not know how it can leave the host cell, migrate, and invade the other host cell. The study of fine structure of both the protozoan and host cell may reveal the answer to these questions. This report adds another example that a protozoan which has been classified in the genus Nosema can produce two spores from one sporont. Foa ( 1924), Paillot ( 1928, 1939), and Steinhaus and Hughes ( 1949) have already reported such cases with other Microsporidia as well as with N. bombycis. The frequency of this type of spore formation should be studied in various species of Microsporidia under controlled conditions. Tissue culture technique provides a suitable method for such studies, __Development of all figures
319
ACICNOWLEDGMENT
I express my thanks to Dr. S. S. Sohi of the Insect Pathology Research Institute, who provided the primary cell cultures used in these experiments. REFERENCES
FOA, A.
1924. Quoted from Paillot (1928). ISHIHARA, R., AND SOHI, S. S. 1966. Infection of ovarian tissue culture of Bombyx mori by Nosema bombycis spores. J. Invertebrate Pathol., 8, 538-540. IWABUCHI, H. 1913. Histological studies on the transovarian infection of Nosema bombycb. Tokyo Sangyo-Koshujo Hokoku (Bull. SangyoKoshujo Tokyo) No. 48, l-65. J. P. 1960. Observations on the KRAMER, emergence of the microsporidian sporoplasm. J. Insect Pathol., 2, 433-439. Kuw, R. R. 1916. Contribution to the study of parasitic protozoa. I. On the structure and of Nosema bombycis Naegeli. life history Bull. Imper. Seric. Expt. Stat. Tokyo, 1, 31-51. Lo&r, J., AND VAVHA, J. 1963. The mode of sporoplasm extrusion in microsporidian spores. Acta Protozool., 1, 81-90. OHSHIMA, K. 1937. On the function of the polar filament of Nosema bombycis. Parasitology, 29, 220-224. oxroar, J. 1912. Zur Kenntnis des Pebrine-Er-
__of Nosema bombycis in tissue culture is 1100X, except Fig. 9 which is 350X.
cells from
ovaries
of Bom.byx
mori.
Magnification
FIG. 1. N. bombycis just after invasion (arrow). Only two nuclei are apparent. FIG. 2. Developing N. bombycti (arrow), with cytoplasm colored deeply blue by Giemsa’s solution. FIG. 3. More advanced stage (arrow) with increased size and original round shape. Nuclei somewhat less compact and irregular in shape. FIG. 4. N. bombycis developed to the elongate form without fission. FIG. 5. Fission of N. bombycis (arrow) after development to the elongate form without fission, FIG. FIG.
6. Elongate from actively dividing in host ccl1 cytoplasm. 7. Steps of typical spore formation. Letters a to f indicate the steps in order of development. FIG. 8. Exceptional method of spore formation where two spores are formed from a sporont. FIG. 9. A host cell infected with spores (sp) and elongate form (nb) of the protozoan as observed under an inverted-phase microscope; N: nucleus of the host cell. FIG. 10. Appearance of infective form (IS) in a host cell. FIG. 11. Infective form (IS) leaving a host cell.
FIG. 12. FIG. 13. FIG. 14.
Infective Infective Infective
form form form
(IS) (IS) (IS)
migrating penetrating developing
from host cell. a new host cell. in a host cell after
invasion.
320
ISHIHARA
Nosema bombycis Naegeli. Arb. Kais. Gesundh., 40, 108-122. PAILLOT, A. 1928. Sur le cycle kvolutif de Nosema bombycis, parasite de la p&brine du Ver B soie. Compt. Rend. Sot. Biol., 99, 81-83. PAILLOT, A. 1939. Le carpocapse dans le region lyonnaise et les regions limitrophes. Ann. Epiphyties Phytogen., 5, 199-211. STEINHAUS, E. A., ASD ~ICJGHES, I(. M. 1949. ragers,
Two newly described species of Microsporidia from the potato tuberworm, Gnorimoschema operculella ( Zeller ) ( Lepidoptera, Gelechiidae). J. Yarasitol., 35, 67-74. STEMPELL, W. 1909. Ueber Nosema bombycis Naegeli. Arch. Protistenk, 16, 281-358. THAGER, W. 1937. The hatching of spores of Nosemu bombycis Naegeli and the partial development of the organism in tissue cultures. J. Parasitol., 23, 226-227.