- Email: [email protected]
Ultrastructure of the midgut of the worker honey bee Apis mellifera heavily infected with Nosema apis
JOURNAL
OF INVERTEBRATE
PATHOLOGY
44, 282-291 (1984)
Ultrastructure of the Midgut of the Worker Honey Bee Apis mellifera Heavily Infected with Nosema apis T. P. LIU Bee Disease
Research
Laboratory.
Agriculture Canada, Research Alberta, TOH OCO, Canada
Station,
Box 29. Beaverlodge,
Received January 3, 1984; accepted April 13, 1984 Midgut epithelial cells from healthy bees possessed numerous mitochondria, strands of endoplasmic reticulum, evenly distributed ribosomes, zymogen granules, and two kinds of lipid inclusions. In heavily infected midguts of honey bees, Apis mellifera, all epithelial cells were observed to be infected with Nosema apis. Cells of the entire midgut were packed with mature spores and, in some cases, mixed with immature stages. Spores were not found among cells of the brush border and basal infolding. Muscle cells and tracheal end cells of the midgut were not infected. The cytoplasm of the infected cell contained a large number of vacuoles, numerous large inclusion bodies, and aggregated ribosomes. Signs of extensive lysis were observed within the heavily infected cells, although the cell membranes were intact. 1984 Academic PIUS. IN. KEY WORDS: Nosema apis Zander; Apis mellifera L.; midgut infection; mature spores; tissue specificity; epithelial cell lysis; tracheal network; inclusion bodies, lipid droplets; zymogen granules; mitochondria; endoplasmic reticulum.
INTRODUCTION
MATERIALS
The most serious effect of Nosema upis on the individual worker honey bee, Apis mellifera, is a reduced longevity (Furgala and Both, 1970). Infected bees live only half as long as noninfected ones (Bailey, 1981): Nosema infection in the honey bee has been shown to cause atrophy of the hypopharynglal glands (Wang, 1969), a tendency to aggravate dysentery (Bailey, 1981), and partial paralysis (Dyess and Wilson, 1978); however, none of these factors contribute directly to the early death of the honey bee. Muresan et al. (1975), based on histochemical observations, suggested that the digestive function of the heavily infected midgut was impaired. Thus, the early death was due to starvation. The present paper reports ultrastructural observations of heavily infected midguts of honey bees as well as those from healthy bees. The significance of these observations are discussed in relation to the functional capacity of infected epithelial cells of midguts.
Specimens of Nosemu-infected worker honey bees were collected from overwintering hives. No infected bees were collected from hives during the summer. The white-colored, swollen, infected midguts (White, 1919) and the straw-colored, healthy midguts (Gochnauer et al., 1979) were dissected and processed for scanning electron microscopy according to the methods described previously (Liu, 1983). The entire surface of the midguts were examined by secondary electron and backscattered electron imaging in the scanning electron microscope in an attempt to find any damage on the surface due to infection. Afterward, the midguts were cut longitudinally into four portions and again coated with gold. The entire length of the four portions was examined. Eleven infected midguts and five noninfected ones were examined. For transmission electron microscopy small pieces of both infected and noninfected midguts were fixed and em282
0022-201 l/84 $1.50 Copyright
0 1984 by Academic
Press, Inc
AND METHODS
Nosema
apis- INFECTED
bedded according to the technique used by Liu and Dixon (1973). Specimens were observed in a Cambridge Steroscan 250 scanning electron microscope at an accelerating voltage of 5-20 kV and in a Philips 201 or 301 transmission electron microscope at an accelerating voltage of 60 kV. RESULTS
The entire midgut of infected and noninfected bees were wrapped in a tracheal network (Fig. 1). The surface of the midgut was distinctly different from that of Malpighian tubules, which were not wrapped with a tracheal network. Underneath the tracheal network, the surface of infected midguts was examined by both secondary and backscattered electron imaging (Fig. 2). Epithelial cells of midguts from healthy bees possessed a dense population of mitochondria, which were especially numerous at the brush border region of the cell (Fig. 3), at the basal region, and within the basal infolding. There were a large number of lipid droplets among the basal infolding and at the basal region of the cell. Large lipid inclusions and a number of zymogen granules were observed at the brush border region of the cell (Fig. 3). Free ribosomes were distributed evenly within the epithelial cells, and strands of endoplasmic reticulum were observed, mainly in the region which surrounds the nucleus. Large inclusion bodies were common features of the healthy epithelial cells. Muscle cells (Fig. 4) and tracheal end cells associated with the midgut were not infected. In the infected midguts, epithelial cells were tightly packed with spores of N. apis (Fig. 5). In three of the infected midguts examined, approximately two-thirds of each midgut (toward the anterior end) was filled with mostly mature spores (Fig. 6). In the posterior portion of each midgut, epithelial cells were packed with mature spores as well as some developmental stages of the spore (Fig. 7). No spores were found among the brush borders (Figs. 5, 8,
MIDGUT
OF Apis mellifera
283
9) or basal infolding of the epithelial cells were free of spores (Fig. 10). There was no breakage of the plasma membrane of the cell either at the base of the brush border (Figs. 8, 9) or the portion of plasma membrane which was free from the brush border (Fig. 9). The infected epithelial cells exhibited extensive lysis, evidenced by the presence of vacuoles, residual glycogen, and aggregated ribosomes (Figs. 8, 9). Numerous membrane-bound inclusion bodies were scattered throughout the cytoplasm (Fig. 11). DISCUSSION
According
to Bailey (1981), spores of N. within the cells of the epithelium of the midgut of adult bees. The present study confirmed that, in the midgut, no other kind of cells were infected. Spores of N. upis were not found in such tissues as Malpighian tubules, fat bodies, or flight muscle (Liu, unpubl.). It appears that N. apis infection is not only tissue specific but also cell specific. This is different from some other Nosema spp. For instance, when Nosema NFW infects the fall webworm, Hyphantria cunia, most tissues are infected in succession in the order midgut, foregut, hindgut, Malpighian tubules, fat bodies, gonads, and muscles (Nordin and Maddox, 1974). The reason for infection specificity of the N. upis is probably due to the fact that the parasite is adapted to the metabolism of the epithelial cells. These cells may require high levels of oxygen for energy metabolism, lipids, and protein synthesis. This hypothesis is supported by the presence of a large number of mitochondria and lipid droplets as well as large inclusions and zymogen granules. The surface of the midgut is covered with a network of tracheae strongly suggesting that the midgut requires high levels of oxygen for metabolism. In the flight muscle of the honey bee glycogen is the predominated energy reserve (Neukirch, 1982), and the honey bee is a synchronous flyer, using apis develop exclusively
T. P. LIU
FIG. Arrow FIG. Bar =
1. A large portion of the infected midgut which is wrapped in a network of trachea (TR). indicates epithelial cells. Bar = 0.2 mm. 2. Tracheal network (TR) viewed by back scattered electron imaging. Tracheal end cell (TC). 10 pm.
Nosema apis- INFECTED
MIDGUT
OF Apis mellifera
FIG. 3. An epithelial cell from noninfected bee contains zymogen granules (small arrow), mitochondria (large arrow), brush border (B), lipid inclusion (L), nucleus (NU), and a large inclusion body (IB). Bar = 10 p,m. FIG. 4. Muscle cells associated with the midgut were not infected. Muscle fiber (MF) and tracheae (Tr). Bar = 4 pm.
286
T. P. LIU
FIG. 5. Infected epithelial cell contains spores (arrow) brush border (B). Bar = 10 km.
Nosema apis- INFECTED
MIDGUT
OF Apis mellifera
FIG. 6. An epithelial cell of the anterior portion of the midgut was fractured open to expose mature spores (SP) of Nosema apis. Bar = 2 p.m. FIG. 7. Epitheliai cells of the posterior portion of the midgut contains mature spores (Sp) and various developmental stages of spores (D). Bar = 4 pm.
287
T. P. LIU
FIG. 8. A portion of the infected epithelial cell -shows extensive lysis. Vacuole (V), spore (Sp), brush border (B), and arrows indicate the aggregated ribosomes. Bar = 1 km. FIG. 9. A portion of the infected cell shows residual glycogen (small arrow), brush border (B), spore (large arrow), and vacuoles of all sizes (V). Bar = 1 pm.
Nosema apis- INFECTED
MIDGUT
OF Apis mellifera
FIG. 10. Spores (arrow) of N. upis were not found in the basal infolding (BS) and basement membrane (B). Tracheae (Tr). Bar = 2 pm. FIG. 11. A portion of infected cell contains a large inclusion body (IB), symbiotom (SB), developmental stages of the spore (DS), and a mature spore (SP). Bar = 1 cm.
289
290
T. P. LIU
only carbohydrate as fuel (Sacktor, 1970). These facts suggest that, besides oxygen and carbohydrate, N. apis requires a larger amount of lipid, protein, and other nutrients for growth and development. Tracheae were absent from the surface of the Malpighian tubules, suggesting that the oxygen requirement is lower than in the midgut. Numerous membrane-bound urate spherules, but not lipid droplets and protein granules, were observed in the cytoplasm of epithelial cells of the Malpighian tubules (Liu, unpubl.). This may be why spores of N. apis were never found within the tubules. Muresan et al. (1975) observed that Nosema infection in the midgut started from the anterior end and spread gradually to the posterior end. The present study supports their view. Observations showed that mature spores were in the anterior twothirds of midguts and the remaining portion contained some immature stages, which suggests that infection started from the anterior end. Muresan et al. (1975) also suggested that cell regeneration ceased due to infection. In the present study noninfected epithelial cells were not observed, and may indicate that either cell regeneration ceased in the infected midgut or infection spread faster than cell regeneration. In heavily infected midguts all infected epithelial cells exhibited a sign of extensive Iysis. However, no lysosomes or autophagic vacuoles were observed. In some insects autophagic vacuoles and lysosomes play an important role in cell death (Scharrer, 1966; Osinchak, 1966; Glitho et al., 1979). In other insects autophagic vacuoles apparently are not involved in cell death (Smith and Nighout, 1982). Lockshin and Beaulaton (1974) have suggested that special lytic enzymes may not be a prerequisite feature of cell death; rather, cell death may result from quantitative changes in rates of turnover of structure molecules such as protein. This suggestion may support the case for the appearance of cell lysis in the heavily infected midgut epithelial
cells of honey bees. This may also explain why the cell plasma membrane was intact when the cytoplasm showed a lysed appearance. Lytic enzyme may indeed not be involved. The lack of zymogen granules and aggregated ribosomes in the heavily infected epithelial cell suggests that digestive enzymes were no longer being secreted. The heavily infected cells may either be dead or dying, which will eventually lead to the early death of the infected worker bees due to starvation, as suggested by Muresan et al. (1975). ACKNOWLEDGMENTS The author thanks Mrs. M. Collins for technical assistance. Thanks are also extended to Mrs. B. Valentine and Mrs. F. Skelton, Research Station, Agriculture Canada, Vancouver, B.C., Canada, for assistance in part of the transmission electron microscopy.
REFERENCES BAILEY, L. 1981. “Honey bee pathology,” Academic Press, New York. DYESS, E. G., AND WILSON, C. A. 1978. A study of the seasonal variations of Nosema apis Zander of honey bees in Mississippi. Amer. Bee J., 118, 3335.
FURGALA, B., AND BOCH, R. 1970. The effect of Fumid&B, Nasemack, and Humatin on Nosema apis. J. Apic.
Res.,
9, 79-85.
GLITHO, I., DELGEQUE, J. P., AND DEZACHAMBRE, J. 1979. Prothoracic gland involution related to moulting hormone level during metamorphosis of Locusta migratoria. J. Insect Physiol. 25, 187- 191. GOCHNAUER,T. A., FURGALA, B., AND SHIMANUKI, H. 1979. Diseases and enemies of the honey bee. In “The Hive and the Honey Bee.” Dadant & Sons, Hamilton, Illinois. Lru, T. P. 1983. Surface structure of spores of Nosema apis Zander as seen in the scanning electron microscope. Amer. Bee J., 123, 810-811. LIU, T. P., AND DIXON, S. E. 1973. Honey bee larval corpora allata: Their fine structure and repressor action. J. Apic. Res., 12, 167-178. LOCKSHIN, R. A., AND BEAULATON, J. 1974. Programmed cell death. Life Sci., 17, 403-410. MURESAN, E., DUCA, D., AND PAPAY, Z. 1975. The study of some histochemical indices of the midgut, healthy and infected with Nosema apis, of the Apis mellifica carpatica bee. In “Proceedings, XXVth Int. Api. Congr.,” pp. 384-385. NEUKIRCH, A. 1982. Dependence of the life span of the honey-bee (Apis mellifera) upon flight perfor-
Nosema
apis- INFECTED
mance and energy consumption. J. Comp.
Physiol.,
144, 35-40.
NORDIN, G. L., AND MADDOX, J. V. 1974. Microsporidia of the fall webworm, Hyphantria cunea L. Identification, distribution, and comparison of Nosema sp, with similar Nosema spp. from other Lepidoptera. J. Invertebr. Puthoi., 24, 1-13. OSINCHAK, J. 1966. Ultrastructural localization of some phosphatases in the prothoracic glands of the insect, Leucophaea maderae. Z. Zeliforsch. Mikrosk.
Anat.,
72, 236-248.
SACKTOR, B. 1970. Regulation of intermediary metabolism, with special reference to the control mechanisms in insect flight muscle. Adv. Insect Physiol., 7, 261-347.
MIDGUT
OF Apis meitifera
291
SCHARRER,B. 1966. Ultrastructural study of the regressing prothoracic glands of blattarian insects. Z. Zellforsch. Mikrosk. Anat., 69, 1-21. SMITH, W. A., AND NIGHOUT, H. F. 1982. Ultrastructural changes accompanying secretion and cell death in the molting glands of an insect (Oncopeltus). Tissue
Celi,
14, 243-252.
WANG, DER-I. 1969. “The effects of behavior, amino acids of the haemolymph, and development of hypopharyngeal glands on Nosema-diseased worker honey bees, Apis mellifera L., on the ability of queens to escape infection by Nosema apis Zander.” Thesis, Univ. of Wisconsin. WHITE, G. F. 1919. “Nosema disease,” USDA Bulletin No. 780, Washington, D.C.
OF INVERTEBRATE
PATHOLOGY
44, 282-291 (1984)
Ultrastructure of the Midgut of the Worker Honey Bee Apis mellifera Heavily Infected with Nosema apis T. P. LIU Bee Disease
Research
Laboratory.
Agriculture Canada, Research Alberta, TOH OCO, Canada
Station,
Box 29. Beaverlodge,
Received January 3, 1984; accepted April 13, 1984 Midgut epithelial cells from healthy bees possessed numerous mitochondria, strands of endoplasmic reticulum, evenly distributed ribosomes, zymogen granules, and two kinds of lipid inclusions. In heavily infected midguts of honey bees, Apis mellifera, all epithelial cells were observed to be infected with Nosema apis. Cells of the entire midgut were packed with mature spores and, in some cases, mixed with immature stages. Spores were not found among cells of the brush border and basal infolding. Muscle cells and tracheal end cells of the midgut were not infected. The cytoplasm of the infected cell contained a large number of vacuoles, numerous large inclusion bodies, and aggregated ribosomes. Signs of extensive lysis were observed within the heavily infected cells, although the cell membranes were intact. 1984 Academic PIUS. IN. KEY WORDS: Nosema apis Zander; Apis mellifera L.; midgut infection; mature spores; tissue specificity; epithelial cell lysis; tracheal network; inclusion bodies, lipid droplets; zymogen granules; mitochondria; endoplasmic reticulum.
INTRODUCTION
MATERIALS
The most serious effect of Nosema upis on the individual worker honey bee, Apis mellifera, is a reduced longevity (Furgala and Both, 1970). Infected bees live only half as long as noninfected ones (Bailey, 1981): Nosema infection in the honey bee has been shown to cause atrophy of the hypopharynglal glands (Wang, 1969), a tendency to aggravate dysentery (Bailey, 1981), and partial paralysis (Dyess and Wilson, 1978); however, none of these factors contribute directly to the early death of the honey bee. Muresan et al. (1975), based on histochemical observations, suggested that the digestive function of the heavily infected midgut was impaired. Thus, the early death was due to starvation. The present paper reports ultrastructural observations of heavily infected midguts of honey bees as well as those from healthy bees. The significance of these observations are discussed in relation to the functional capacity of infected epithelial cells of midguts.
Specimens of Nosemu-infected worker honey bees were collected from overwintering hives. No infected bees were collected from hives during the summer. The white-colored, swollen, infected midguts (White, 1919) and the straw-colored, healthy midguts (Gochnauer et al., 1979) were dissected and processed for scanning electron microscopy according to the methods described previously (Liu, 1983). The entire surface of the midguts were examined by secondary electron and backscattered electron imaging in the scanning electron microscope in an attempt to find any damage on the surface due to infection. Afterward, the midguts were cut longitudinally into four portions and again coated with gold. The entire length of the four portions was examined. Eleven infected midguts and five noninfected ones were examined. For transmission electron microscopy small pieces of both infected and noninfected midguts were fixed and em282
0022-201 l/84 $1.50 Copyright
0 1984 by Academic
Press, Inc
AND METHODS
Nosema
apis- INFECTED
bedded according to the technique used by Liu and Dixon (1973). Specimens were observed in a Cambridge Steroscan 250 scanning electron microscope at an accelerating voltage of 5-20 kV and in a Philips 201 or 301 transmission electron microscope at an accelerating voltage of 60 kV. RESULTS
The entire midgut of infected and noninfected bees were wrapped in a tracheal network (Fig. 1). The surface of the midgut was distinctly different from that of Malpighian tubules, which were not wrapped with a tracheal network. Underneath the tracheal network, the surface of infected midguts was examined by both secondary and backscattered electron imaging (Fig. 2). Epithelial cells of midguts from healthy bees possessed a dense population of mitochondria, which were especially numerous at the brush border region of the cell (Fig. 3), at the basal region, and within the basal infolding. There were a large number of lipid droplets among the basal infolding and at the basal region of the cell. Large lipid inclusions and a number of zymogen granules were observed at the brush border region of the cell (Fig. 3). Free ribosomes were distributed evenly within the epithelial cells, and strands of endoplasmic reticulum were observed, mainly in the region which surrounds the nucleus. Large inclusion bodies were common features of the healthy epithelial cells. Muscle cells (Fig. 4) and tracheal end cells associated with the midgut were not infected. In the infected midguts, epithelial cells were tightly packed with spores of N. apis (Fig. 5). In three of the infected midguts examined, approximately two-thirds of each midgut (toward the anterior end) was filled with mostly mature spores (Fig. 6). In the posterior portion of each midgut, epithelial cells were packed with mature spores as well as some developmental stages of the spore (Fig. 7). No spores were found among the brush borders (Figs. 5, 8,
MIDGUT
OF Apis mellifera
283
9) or basal infolding of the epithelial cells were free of spores (Fig. 10). There was no breakage of the plasma membrane of the cell either at the base of the brush border (Figs. 8, 9) or the portion of plasma membrane which was free from the brush border (Fig. 9). The infected epithelial cells exhibited extensive lysis, evidenced by the presence of vacuoles, residual glycogen, and aggregated ribosomes (Figs. 8, 9). Numerous membrane-bound inclusion bodies were scattered throughout the cytoplasm (Fig. 11). DISCUSSION
According
to Bailey (1981), spores of N. within the cells of the epithelium of the midgut of adult bees. The present study confirmed that, in the midgut, no other kind of cells were infected. Spores of N. upis were not found in such tissues as Malpighian tubules, fat bodies, or flight muscle (Liu, unpubl.). It appears that N. apis infection is not only tissue specific but also cell specific. This is different from some other Nosema spp. For instance, when Nosema NFW infects the fall webworm, Hyphantria cunia, most tissues are infected in succession in the order midgut, foregut, hindgut, Malpighian tubules, fat bodies, gonads, and muscles (Nordin and Maddox, 1974). The reason for infection specificity of the N. upis is probably due to the fact that the parasite is adapted to the metabolism of the epithelial cells. These cells may require high levels of oxygen for energy metabolism, lipids, and protein synthesis. This hypothesis is supported by the presence of a large number of mitochondria and lipid droplets as well as large inclusions and zymogen granules. The surface of the midgut is covered with a network of tracheae strongly suggesting that the midgut requires high levels of oxygen for metabolism. In the flight muscle of the honey bee glycogen is the predominated energy reserve (Neukirch, 1982), and the honey bee is a synchronous flyer, using apis develop exclusively
T. P. LIU
FIG. Arrow FIG. Bar =
1. A large portion of the infected midgut which is wrapped in a network of trachea (TR). indicates epithelial cells. Bar = 0.2 mm. 2. Tracheal network (TR) viewed by back scattered electron imaging. Tracheal end cell (TC). 10 pm.
Nosema apis- INFECTED
MIDGUT
OF Apis mellifera
FIG. 3. An epithelial cell from noninfected bee contains zymogen granules (small arrow), mitochondria (large arrow), brush border (B), lipid inclusion (L), nucleus (NU), and a large inclusion body (IB). Bar = 10 p,m. FIG. 4. Muscle cells associated with the midgut were not infected. Muscle fiber (MF) and tracheae (Tr). Bar = 4 pm.
286
T. P. LIU
FIG. 5. Infected epithelial cell contains spores (arrow) brush border (B). Bar = 10 km.
Nosema apis- INFECTED
MIDGUT
OF Apis mellifera
FIG. 6. An epithelial cell of the anterior portion of the midgut was fractured open to expose mature spores (SP) of Nosema apis. Bar = 2 p.m. FIG. 7. Epitheliai cells of the posterior portion of the midgut contains mature spores (Sp) and various developmental stages of spores (D). Bar = 4 pm.
287
T. P. LIU
FIG. 8. A portion of the infected epithelial cell -shows extensive lysis. Vacuole (V), spore (Sp), brush border (B), and arrows indicate the aggregated ribosomes. Bar = 1 km. FIG. 9. A portion of the infected cell shows residual glycogen (small arrow), brush border (B), spore (large arrow), and vacuoles of all sizes (V). Bar = 1 pm.
Nosema apis- INFECTED
MIDGUT
OF Apis mellifera
FIG. 10. Spores (arrow) of N. upis were not found in the basal infolding (BS) and basement membrane (B). Tracheae (Tr). Bar = 2 pm. FIG. 11. A portion of infected cell contains a large inclusion body (IB), symbiotom (SB), developmental stages of the spore (DS), and a mature spore (SP). Bar = 1 cm.
289
290
T. P. LIU
only carbohydrate as fuel (Sacktor, 1970). These facts suggest that, besides oxygen and carbohydrate, N. apis requires a larger amount of lipid, protein, and other nutrients for growth and development. Tracheae were absent from the surface of the Malpighian tubules, suggesting that the oxygen requirement is lower than in the midgut. Numerous membrane-bound urate spherules, but not lipid droplets and protein granules, were observed in the cytoplasm of epithelial cells of the Malpighian tubules (Liu, unpubl.). This may be why spores of N. apis were never found within the tubules. Muresan et al. (1975) observed that Nosema infection in the midgut started from the anterior end and spread gradually to the posterior end. The present study supports their view. Observations showed that mature spores were in the anterior twothirds of midguts and the remaining portion contained some immature stages, which suggests that infection started from the anterior end. Muresan et al. (1975) also suggested that cell regeneration ceased due to infection. In the present study noninfected epithelial cells were not observed, and may indicate that either cell regeneration ceased in the infected midgut or infection spread faster than cell regeneration. In heavily infected midguts all infected epithelial cells exhibited a sign of extensive Iysis. However, no lysosomes or autophagic vacuoles were observed. In some insects autophagic vacuoles and lysosomes play an important role in cell death (Scharrer, 1966; Osinchak, 1966; Glitho et al., 1979). In other insects autophagic vacuoles apparently are not involved in cell death (Smith and Nighout, 1982). Lockshin and Beaulaton (1974) have suggested that special lytic enzymes may not be a prerequisite feature of cell death; rather, cell death may result from quantitative changes in rates of turnover of structure molecules such as protein. This suggestion may support the case for the appearance of cell lysis in the heavily infected midgut epithelial
cells of honey bees. This may also explain why the cell plasma membrane was intact when the cytoplasm showed a lysed appearance. Lytic enzyme may indeed not be involved. The lack of zymogen granules and aggregated ribosomes in the heavily infected epithelial cell suggests that digestive enzymes were no longer being secreted. The heavily infected cells may either be dead or dying, which will eventually lead to the early death of the infected worker bees due to starvation, as suggested by Muresan et al. (1975). ACKNOWLEDGMENTS The author thanks Mrs. M. Collins for technical assistance. Thanks are also extended to Mrs. B. Valentine and Mrs. F. Skelton, Research Station, Agriculture Canada, Vancouver, B.C., Canada, for assistance in part of the transmission electron microscopy.
REFERENCES BAILEY, L. 1981. “Honey bee pathology,” Academic Press, New York. DYESS, E. G., AND WILSON, C. A. 1978. A study of the seasonal variations of Nosema apis Zander of honey bees in Mississippi. Amer. Bee J., 118, 3335.
FURGALA, B., AND BOCH, R. 1970. The effect of Fumid&B, Nasemack, and Humatin on Nosema apis. J. Apic.
Res.,
9, 79-85.
GLITHO, I., DELGEQUE, J. P., AND DEZACHAMBRE, J. 1979. Prothoracic gland involution related to moulting hormone level during metamorphosis of Locusta migratoria. J. Insect Physiol. 25, 187- 191. GOCHNAUER,T. A., FURGALA, B., AND SHIMANUKI, H. 1979. Diseases and enemies of the honey bee. In “The Hive and the Honey Bee.” Dadant & Sons, Hamilton, Illinois. Lru, T. P. 1983. Surface structure of spores of Nosema apis Zander as seen in the scanning electron microscope. Amer. Bee J., 123, 810-811. LIU, T. P., AND DIXON, S. E. 1973. Honey bee larval corpora allata: Their fine structure and repressor action. J. Apic. Res., 12, 167-178. LOCKSHIN, R. A., AND BEAULATON, J. 1974. Programmed cell death. Life Sci., 17, 403-410. MURESAN, E., DUCA, D., AND PAPAY, Z. 1975. The study of some histochemical indices of the midgut, healthy and infected with Nosema apis, of the Apis mellifica carpatica bee. In “Proceedings, XXVth Int. Api. Congr.,” pp. 384-385. NEUKIRCH, A. 1982. Dependence of the life span of the honey-bee (Apis mellifera) upon flight perfor-
Nosema
apis- INFECTED
mance and energy consumption. J. Comp.
Physiol.,
144, 35-40.
NORDIN, G. L., AND MADDOX, J. V. 1974. Microsporidia of the fall webworm, Hyphantria cunea L. Identification, distribution, and comparison of Nosema sp, with similar Nosema spp. from other Lepidoptera. J. Invertebr. Puthoi., 24, 1-13. OSINCHAK, J. 1966. Ultrastructural localization of some phosphatases in the prothoracic glands of the insect, Leucophaea maderae. Z. Zeliforsch. Mikrosk.
Anat.,
72, 236-248.
SACKTOR, B. 1970. Regulation of intermediary metabolism, with special reference to the control mechanisms in insect flight muscle. Adv. Insect Physiol., 7, 261-347.
MIDGUT
OF Apis meitifera
291
SCHARRER,B. 1966. Ultrastructural study of the regressing prothoracic glands of blattarian insects. Z. Zellforsch. Mikrosk. Anat., 69, 1-21. SMITH, W. A., AND NIGHOUT, H. F. 1982. Ultrastructural changes accompanying secretion and cell death in the molting glands of an insect (Oncopeltus). Tissue
Celi,
14, 243-252.
WANG, DER-I. 1969. “The effects of behavior, amino acids of the haemolymph, and development of hypopharyngeal glands on Nosema-diseased worker honey bees, Apis mellifera L., on the ability of queens to escape infection by Nosema apis Zander.” Thesis, Univ. of Wisconsin. WHITE, G. F. 1919. “Nosema disease,” USDA Bulletin No. 780, Washington, D.C.