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In vitro phagocytosis of Nosema apis spores by honey-bee hemocytes
JOURNAL
OF
INVERTEBRATE
In
9,
PATHOLOGY
Vitro
387389
Phagocytosis Honey-Bee
MARTHA
GILLIAM~
Entomology Research U.S. Department
of
Nosema apis HemocytedJ2
AND HACHIRO
Spores
by
SHIMANUKI~
Division, Agricultural Research Service of Agriculture, Lammie, Wyoming
Accepted Phagocytozed Nosema apis spores to rupture. This phenomenon and the ensuing death of the insect. mellifera,
(1967)
September
13,
1966
caused hemocytes may be responsible
of
the honey bee, for the weakened
Apis state
ejected with feces to contaminate the food or water of other bees, thus transNosema disease of the honey bee, mitting the protozoan. Apis mellifera, is caused by a microscopic A gradual weakening of infected insects single-celled protozoan, Nosema apis, beis quite characteristic; diseased bees are longing to the order Microsporidia. The unable to fly without alighting frequently. life cycle of this organism has not yet been Also the stomach is swollen, and becomes elucidated. However, in one stage of its white in color because of the spores. The development, Nosema apis forms highly disease is usually diagnosed by removing resistant spores which refract light so that, the abdomen with forceps and pulling out even without staining, they can be seen the digestive tract; the gut contents are clearly under the microscope. then smeared upon a slide and observed The pathogen is transmitted through under the microscope for the presence ot food, water, or equipment contaminated spores. with Nosema spores. First the young paraProfessor Zander ( 1909), who discov sites grow and multiply within the stomered the causative organism of nosema ach; then they enter the epithelial cells disease, stated that the midgut of the bee lining the midgut (Grout, 1949). When is the main site of this parasite. However, the walls of the epithelial cells break, the Steche ( 1960) found Nosema apis in the spores are liberated. They can now be ovaries and mandibular glands. Also Soko’ The data contained herein constituted a part lov and Grobov (1963) found Nosemu of a thesis submitted by the senior author in parspores in the hemolymph, pharyngeal tial fulfillment of the requirements for the degree gland, and thoracic gland of infected bees of Master of Science, University of Wyoming. and noted that Nosemu spores introducetl Published with approval of the Director, Wyoming Agricultural Experiment Station, as with hemolymph into healthy bees caused Journal Article 302. typical development of the disease; there” Present address: Department of Microbiology, fore they postulated that the organism University of Arizona, Tucson. causing nosema disease can probably go ’ Present address: Bee Disease Laboratory, Agrifrom the hemolymph into the gut as well cultural Research Center, Beltsville, Maryland. 387
388
GIL,LIAM
AND
as from the gut into the hemolymph. On the other hand, Miiller ( 1925) observed no differences between the blood corpuscles of healthy bees and those of bees infected with Nosemu. No data have been reported on the phagocytic qualities of honey-bee hemocytes, but while performing total hemocyte counts (THC ) on honey bees, we noticed that bees infected with Nosema had fewer blood cells than healthy bees. This investigation was therefore made to determine whether the hemocytes are able to engulf Nosemu spores and to observe the effect of the phagocytosis upon the blood cells. MATERIALS
AND
METHODS
Comparisons were made of the average THC of healthy 5-day-old larvae and 5day-old larvae from colonies infected with Nosema apk (Five-day-old larvae were used to avoid any variation in the THC due to the age of the insect. ) To obtain hemolymph for the THC, larvae were punctured with a sterile hypodermic needle. Then the hemolymph which exuded from the wound was drawn to the 0.5 mark of a Thoma-white-cell diluting pipette and diluted to the 11 mark with Toisson’s fluid (1.0 g sodium chloride, 8.0 g sodium sulfate, 30 ml glycerin, 15 mg crystal violet, and 160 ml distilled water). After thorough mixing for 2 minutes, we discarded the first three drops of the fluid, and the fourth was used for the count. A Spencer bright-line hemacytometer with improved Neubauer ruling was used, the cells in the four comers and central squares were counted, and the sum was multiplied by 40 to give the number of cells/mm3. If the cells were unevenly distributed, the sample was discarded. The average THC reported was based upon five individual samples. TO test the effect of Nosema spores on phagocytosis, we placed 0.01 cc of larval
SHIMANUKI
hemolymph in a depression slide, and 0.02 cc of a lO*/ml suspension of Nosema apis spores was added. The slide was observed at a magnification of 430 X for 45 minutes at room temperature. The final pH of the suspension of hemolymph and spores ranged from 6.8 to 7.2. The pH of larval blood is about 6.8 (Bishop, 1923). RESULTS
AND
DISCUSSION
The THC of the S-day-old larvae from healthy colonies was higher than that of the same age larvae from colonies infected by Nosemu apis (Table 1). Also spores of Nosema apis were found in the hemolymph of larvae from the diseased colonies. When the suspension of Nosema apti spores was added to the hemolymph, the hemocytes remained relatively stationary (Fig. 1). However, spores appeared to be attracted to the blood cells by some sort of chemotactic action and entered through the cell walls. No pseudopods were observed (Fig. IA-H). As the blood cells became graduahy filled with spores, a characteristic invagination of the cell occurred (Fig. 11). The hemocytes then ruptured because of the quantity of spores ingested, and the cell contents spilled out ( Fig. 1J-L). This entire process took about 45 minutes. All types of blood cells appeared to be affected. Thus Nosema spores in sufficient quantities tended to destroy the hemocytes. TABLE THC
Condition
1
OF LARVAE FROM DISEASED AND HEALTHY COLONIES
of colony
Average
Healthy Healthy Nosema-infected Nosema-infected LI Based
on five
10,280 10,040 3,907 5,480 individual
samples.
THCa
PHAGOCYTOZED
FIG.
1. Phagocytosis
of
Nusema apis spores
This destruction of blood cells would eventually weaken the insect, and death would result. The process may therefore possibly explain the symptoms of honey bees infected with Nosenza apis. The THC proved a good diagnostic test for Nosema since that of infected bees was lower than that of healthy bees. The spores could also be observed in the hemolymph. REFERENCES
G. H. 1923. Body fluid of the honey bee larvae. I. Osmotic pressure, specific gravity, pH, 0, capacity, CO, capacity, and buffer value, and their changes with larval activity
BISHOP,
upis
Nosemu
389
SPORES
by
honey-bee
hemocytes.
and metamorphosis. J. Biol. Chem.. 58, 643565. GHOUT, R. A. 1949. “The Hive and the Hone) Bee,” Dadant and Sons, 652 pp. Hamilton, Illinois. M~~LLEI~, K. l92*5. Wber die korpuskutiiren Elemente dcr Blutfliissigkeit bei der erwachsenen Honigbiene (Avis mellifica L. ) Edanger
Jahrb. Bienenkunde, SOKOLOV,
v.
P., AND
3, t5-27.
&OBOV,
spores
0.
in the hemolymph coflstvo, 7, 39. STECHE, W. 1960. dtiologie Nosematose der Honigbiene,
F.
l%%.
of the bee. und
Therapie
~OXWXl
Pchdotier
Z. Biencnfwwh..
5, 39-52. ZAPI‘DER, E. 1909. Tierische heitserreger Bei der ncxtg., 24, l-47-150,
Parasiten als KrankBiene. Leipzigrr Hick164-166.
OF
INVERTEBRATE
In
9,
PATHOLOGY
Vitro
387389
Phagocytosis Honey-Bee
MARTHA
GILLIAM~
Entomology Research U.S. Department
of
Nosema apis HemocytedJ2
AND HACHIRO
Spores
by
SHIMANUKI~
Division, Agricultural Research Service of Agriculture, Lammie, Wyoming
Accepted Phagocytozed Nosema apis spores to rupture. This phenomenon and the ensuing death of the insect. mellifera,
(1967)
September
13,
1966
caused hemocytes may be responsible
of
the honey bee, for the weakened
Apis state
ejected with feces to contaminate the food or water of other bees, thus transNosema disease of the honey bee, mitting the protozoan. Apis mellifera, is caused by a microscopic A gradual weakening of infected insects single-celled protozoan, Nosema apis, beis quite characteristic; diseased bees are longing to the order Microsporidia. The unable to fly without alighting frequently. life cycle of this organism has not yet been Also the stomach is swollen, and becomes elucidated. However, in one stage of its white in color because of the spores. The development, Nosema apis forms highly disease is usually diagnosed by removing resistant spores which refract light so that, the abdomen with forceps and pulling out even without staining, they can be seen the digestive tract; the gut contents are clearly under the microscope. then smeared upon a slide and observed The pathogen is transmitted through under the microscope for the presence ot food, water, or equipment contaminated spores. with Nosema spores. First the young paraProfessor Zander ( 1909), who discov sites grow and multiply within the stomered the causative organism of nosema ach; then they enter the epithelial cells disease, stated that the midgut of the bee lining the midgut (Grout, 1949). When is the main site of this parasite. However, the walls of the epithelial cells break, the Steche ( 1960) found Nosema apis in the spores are liberated. They can now be ovaries and mandibular glands. Also Soko’ The data contained herein constituted a part lov and Grobov (1963) found Nosemu of a thesis submitted by the senior author in parspores in the hemolymph, pharyngeal tial fulfillment of the requirements for the degree gland, and thoracic gland of infected bees of Master of Science, University of Wyoming. and noted that Nosemu spores introducetl Published with approval of the Director, Wyoming Agricultural Experiment Station, as with hemolymph into healthy bees caused Journal Article 302. typical development of the disease; there” Present address: Department of Microbiology, fore they postulated that the organism University of Arizona, Tucson. causing nosema disease can probably go ’ Present address: Bee Disease Laboratory, Agrifrom the hemolymph into the gut as well cultural Research Center, Beltsville, Maryland. 387
388
GIL,LIAM
AND
as from the gut into the hemolymph. On the other hand, Miiller ( 1925) observed no differences between the blood corpuscles of healthy bees and those of bees infected with Nosemu. No data have been reported on the phagocytic qualities of honey-bee hemocytes, but while performing total hemocyte counts (THC ) on honey bees, we noticed that bees infected with Nosema had fewer blood cells than healthy bees. This investigation was therefore made to determine whether the hemocytes are able to engulf Nosemu spores and to observe the effect of the phagocytosis upon the blood cells. MATERIALS
AND
METHODS
Comparisons were made of the average THC of healthy 5-day-old larvae and 5day-old larvae from colonies infected with Nosema apk (Five-day-old larvae were used to avoid any variation in the THC due to the age of the insect. ) To obtain hemolymph for the THC, larvae were punctured with a sterile hypodermic needle. Then the hemolymph which exuded from the wound was drawn to the 0.5 mark of a Thoma-white-cell diluting pipette and diluted to the 11 mark with Toisson’s fluid (1.0 g sodium chloride, 8.0 g sodium sulfate, 30 ml glycerin, 15 mg crystal violet, and 160 ml distilled water). After thorough mixing for 2 minutes, we discarded the first three drops of the fluid, and the fourth was used for the count. A Spencer bright-line hemacytometer with improved Neubauer ruling was used, the cells in the four comers and central squares were counted, and the sum was multiplied by 40 to give the number of cells/mm3. If the cells were unevenly distributed, the sample was discarded. The average THC reported was based upon five individual samples. TO test the effect of Nosema spores on phagocytosis, we placed 0.01 cc of larval
SHIMANUKI
hemolymph in a depression slide, and 0.02 cc of a lO*/ml suspension of Nosema apis spores was added. The slide was observed at a magnification of 430 X for 45 minutes at room temperature. The final pH of the suspension of hemolymph and spores ranged from 6.8 to 7.2. The pH of larval blood is about 6.8 (Bishop, 1923). RESULTS
AND
DISCUSSION
The THC of the S-day-old larvae from healthy colonies was higher than that of the same age larvae from colonies infected by Nosemu apis (Table 1). Also spores of Nosema apis were found in the hemolymph of larvae from the diseased colonies. When the suspension of Nosema apti spores was added to the hemolymph, the hemocytes remained relatively stationary (Fig. 1). However, spores appeared to be attracted to the blood cells by some sort of chemotactic action and entered through the cell walls. No pseudopods were observed (Fig. IA-H). As the blood cells became graduahy filled with spores, a characteristic invagination of the cell occurred (Fig. 11). The hemocytes then ruptured because of the quantity of spores ingested, and the cell contents spilled out ( Fig. 1J-L). This entire process took about 45 minutes. All types of blood cells appeared to be affected. Thus Nosema spores in sufficient quantities tended to destroy the hemocytes. TABLE THC
Condition
1
OF LARVAE FROM DISEASED AND HEALTHY COLONIES
of colony
Average
Healthy Healthy Nosema-infected Nosema-infected LI Based
on five
10,280 10,040 3,907 5,480 individual
samples.
THCa
PHAGOCYTOZED
FIG.
1. Phagocytosis
of
Nusema apis spores
This destruction of blood cells would eventually weaken the insect, and death would result. The process may therefore possibly explain the symptoms of honey bees infected with Nosenza apis. The THC proved a good diagnostic test for Nosema since that of infected bees was lower than that of healthy bees. The spores could also be observed in the hemolymph. REFERENCES
G. H. 1923. Body fluid of the honey bee larvae. I. Osmotic pressure, specific gravity, pH, 0, capacity, CO, capacity, and buffer value, and their changes with larval activity
BISHOP,
upis
Nosemu
389
SPORES
by
honey-bee
hemocytes.
and metamorphosis. J. Biol. Chem.. 58, 643565. GHOUT, R. A. 1949. “The Hive and the Hone) Bee,” Dadant and Sons, 652 pp. Hamilton, Illinois. M~~LLEI~, K. l92*5. Wber die korpuskutiiren Elemente dcr Blutfliissigkeit bei der erwachsenen Honigbiene (Avis mellifica L. ) Edanger
Jahrb. Bienenkunde, SOKOLOV,
v.
P., AND
3, t5-27.
&OBOV,
spores
0.
in the hemolymph coflstvo, 7, 39. STECHE, W. 1960. dtiologie Nosematose der Honigbiene,
F.
l%%.
of the bee. und
Therapie
~OXWXl
Pchdotier
Z. Biencnfwwh..
5, 39-52. ZAPI‘DER, E. 1909. Tierische heitserreger Bei der ncxtg., 24, l-47-150,
Parasiten als KrankBiene. Leipzigrr Hick164-166.