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Unique structures in the insect granular hemocytes
© 1971 by Academic Press, Inc.
646
j. ULTRASTRUCTURERESEARCH36, 646--658 (197l)
Unique Structures in the Insect Gronulor Hemocytes MARTIN HAGOPIAN Delzartment of Pathology, New York Medical College, New York, New York 10029 Received November 23, 1970, and in revised form February 4, 1971 The ultrastructural studies of the cockroach nymph granular hemocytes show structures in the cytoplasm that have never been described. Some of the inclusions have structural characteristics suggestive of melanosomes and premelanosomes, plastids, and microbodies. It is particularly interesting that some granules contain microtubules (250 A in diameter) which display central elements. The central elements are approximately 50 A in diameter and reveal 8 spokes radiating from them. In addition the granular hemocytes show marginal bundles of microtubules which are always in close proximity to the cell membrane. These tubules lack the 50 it central elements. The findings are discussed in terms of previous known functions and histochemistry of insect hemocytes. Some of the known functions of insect hemocytes are phagocytosis, encapsulation, melanization, wound healing, coagulation, and possible transformation into other tissues. Also hemocytes have been reported to lyse among differentiating tissues and appear to have a nutritive effect [see Jones (10) for review]. There are several different types of hemocytes, but little is known about their fine structure. Previous studies of these cells lack the ultrastructural morphology that is helpful in determining cellular function. Therefore it would be significant to further elucidate the role of hemocytes by such morphological studies. The purpose of the present study is to describe some cytoplasmic structures and the various stages of development of the highly ordered granules in the cockroach nymph granular hemocytes. The findings are discussed in terms of previous known functions and histochemistry of insect hemocytes.
MATERIALS AND METHODS Femoral segments from the metathoracic leg (hind leg) of the freshly molted cockroach nymph, Leucophaea maderae, were removed and placed into phosphate-buffered 5 % glutaraldehyde at pH 7.4 for 3 hours (room temperature). While in the fixative the exoskeleton was perforated to ensure proper fixation. The tissue was then rinsed in 0.2 M phosphate buffer for 15 hours and postfixed in 2% osmium tetroxide (phosphate-buffered at pH 7.4) for 1 hour at room temperature. Dehydration in various grades of acetone and embedding in Araldite followed. Thin sections were doubly stained with uranyl acetate and lead citrate and were examined in a Philips EM 200 or a Siemens 1A electron microscope.
UNIQUE STRUCTURESIN INSECT HEMOCYTES
647
RESULTS The femoral cuticle with the underlying epidermal cells of the cockroach nymph enclosed the extensor and flexor muscles, nerves, tracheae, tracheoles, and the hemocoel which contains hemolymph and hemocytes. In this open circulatory system the granular hemocytes (perhaps several types) in the hemolymph are conspicuous by their general elliptical appearance, large nucleus, and prominent cytoplasmic granules (Fig. 1). The cytoplasm also contains mitochondria (small and few in number), extensive Golgi regions, rough and smooth endoplasmic reticulum, free ribosomes, lysosome-like bodies, and marginal bundles of microtubules. Glycogen granules appeared to be absent. Although evidence of mitosis has never been noted, these circulating blood cells are present in considerable numbers in the freshly molted n y m p h (within an hour after ecdysis).
Melanosome-like and premelanosome-like granules The most abundant type of granules are the membrane-bounded, roughly spherical, opaque inclusions which average about 1-2 # in diameter (Fig. 2). No inner structure of these inclusions may be resolved even at high magnifications. These dense inclusions resemble vertebrate melanosomes. Other granules have a highly organized substructure (Figs. 3-7) and are reminiscent of the vertebrate premelanosomes (2, 15, 19). Figure 3 shows that the earliest stage of the premelanosome-like inclusions seems to be derived from the Golgi region whereas Figs. 4-7 demonstrate the deposition of membranes inside these granules at various stages of morphogenesis. In Fig. 8 the internal configurations show their concentric arrangement. Furthermore, certain planes of sections of these granules reveal that the internal configurations are microtubules which measure about 250 A in diameter (Figs. 9 and 10). Of particular interest (at a high magnification and at the proper orientation) are the centers of the microtubules. The centers suggest micro-microtubules even though no evidence is presented concerning the three-dimensional structure of the central element. These small tubules are approximately 50 A in diameter and reveal 8 spokes radiating from them (Fig. 11, arrowhead). Perhaps because the central element is so small, it is demonstrated in very FIO. 1. An electron micrograph of a granular hemocyte. It is characterized by its elliptical shape, large nucleus, and prominent dense granules, x 20 000. FIG. 2. A micrograph of a melanin-like granule. It is membrane-bounded and has a dense unstructured matrix, x 40 000. FIG. 3. Portion of a granular hemocyte showing the possible derivation of the early premelanosomelike granules (P) from the Golgi region (G). MG, melanin-like granule; MT, microtubules, x 30 000. FIo. 4. An early premelanosome-like granule displaying the deposition of some internal encircling membranes (arrowhead). x 50 000. FIG. 5. View of a premelanosome-like body demonstrating the internal membrane arrangement. This symmetrical orientation resembles the vertebrate premelanosome, x 40 000.
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650
HAGOPIAN
few cases. Also it is noted in Fig. 11 that if the orientation is not exactly right neither the micro-microtubules nor the spokes radiating f r o m them are visible. In the same micrograph the outer m e m b r a n e defining the premelanosome-like granule exhibits a typical unit membrane. At the different stages of granular morphogenesis the density of the premelanosome-like granules increase until they ultimately become opaque and unstructured. On occasion the whole granular hemocyte laden with dense granules appears to be disintegrating. However the possibility of fixation damage can not be ignored even t h o u g h surrounding cells seem well preserved.
The plastid-like structures These elongated structures measure a b o u t 1.0-1.5 # in length and are approximately 0.2-0.4 # in width. A l t h o u g h they are b o u n d e d by a single membrane, their overall appearance resembles chloroplasts of lower plant forms which are always defined by a double m e m b r a n e (3). Within, the internal membranes generally extend f r o m one end of the long axis to the other end in parallel arrays (Figs. 12 and 13). Furthermore the membranes vary in n u m b e r and often show a localized dense spot at one end (Fig. 12).
Microbody-like granules The microbody-like granules are often encountered in the granular hemocytes. These granules are b o u n d e d by a unit membrane, are spherical in shape, and measure 0.5-1.0 # in diameter (Fig. 14). The finely granular matrix is fairly h o m o g e n e o u s and has a moderate electron opacity. Because of the finer granular matrix, these bodies are distinguishable f r o m early stages of premelanosome-like granules. N o crystalline inclusions have ever been observed in the matrices of these bodies.
Marginal bundles of rnicrotubules Marginal bands, consisting of bundles of microtubules, are seen in the granular hemocytes. The microtubules are arranged in loosely assembled bundles and are Fro. 6. A group of premelanosome-like granules at various orientations. × 38 000. F:G. 7. A more advanced stage of premelanosome-like granules than that in Fig. 6. The granules have more internal membranes and are denser, x 31 000. FIG. 8. Another plane of section of the premelanosome-like granules showing the concentric arrangement of the internal membranes. × 38 000. FIG. 9. At a higher magnification and at the proper orientation the internal configuration may be seen to be microtubules which measure approximately 250/~ in diameter, x 63 000. F:G. 10. Another view of a premelanosome-like granule illustrating the internal microtubules. x 54 000. FIG. 11. A high-power micrograph of a premelanosome-like granule. Especially note the central elements, which measure about 50 ~ in diameter, in the center of the microtubules. These small tubules have 8 spokes radiating from them (arrowhead). The microtubules and central elements may function as a passageway for the transport of metabolites in the granule. Also note the unit membrane limiting the granule (1). x 240 000.
UNIQUE STRUCTURES IN INSECT HEMOCYTES
651
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UNIQUE STRUCTURES IN INSECT HEMOCYTES
653
654
HAGOPIAN
FIGS. 12 and 13. Examples of plastid-like structures (PS). These membrane-containing components are bounded by a single membrane. The internal membranes, which vary in number, generally extend from one end of the long axis to the other end in parallel arrays, and occasionally a dense spot (arrowhead) is found at one end. The function of these inclusions remains obscure. MT, microtubules. Fig. 12, × 40 000. Fig. 13, x 42 000.
UNIQUE STRUCTURES IN INSECT HEMOCYTES
655
FIG. 14. A microbody-like inclusion. These unit membrane-bounded granules are spherical in shape and measure 0.5-1.0/~ in diameter. The finely granular matrix has a moderate electron opacity. x 72 000. Fro, 15. Marginal bundles of microtubules about 250/~ in diameter (MT) are loosely assembled in close proximity to the granular hemocyte plasma membrane. In cross sections these microtubules do not have the centrally located 50/~ micro-microtubules that are found in some of the premelanosomelike granules. The marginal tubules appear to encircle the cell and are suggestive of a cytoskeletal function. H, precipitated hemolymph, x 50 000.
656
HAGOPIAN
always in close proximity to the cell membrane. The tubules are never connected to the surface of the cell or to each other and seem to encircle the disk-shaped cell. In certain planes of section they manifest themselves in cross or slightly tangential orientation at the poles of the cells (Figs. 3 and 13). Other planes of section reveal marginal arrangements of longitudinally oriented microtubules (Fig. 15). In cross sections the tubules vary from 25 to 35 per bundle, are surrounded by a halo, and are about 250 A in diameter. These microtubules lack the centrally located 50 A micro-microtubules that are seen in the microtubules of some granules. DISCUSSION The granular hemocytes of the cockroach nymph reveal well-preserved structures in their cytoplasm that have never been described. Many of the highly organized granules in the multifunctional, circulating blood cells appear to be in various stages of morphogenesis. At times some of these developing granules and some of the other inclusions show structural characteristics suggestive of melanosomes and premelanosomes, plastids, and microbodies (peroxisomes). Such findings add supportive morphological evidence to known functions and histochemistry of hemocytes [see Jones (10) for review] and also present additional information. The most common and most striking inclusions are those that resemble the melanosomes and premelanosomes found in vertebrate melanocytes and melanomas (2, 15, 19). In support of this resemblance Kaplan (11) has reported that in some specific cases hemocytes of insects may be involved in the melanization of encapsulated material. Salt (18) has shown that hemocytes in the developmental stages of Tenebrio envelop and melanize eggs of an ichneumon fly. Also tyrosinase has been found as a chemical constituent of hemocytes by Jones (9), Vercauteren and Aerts (21), Rizki and Rizki (17), and Kawase (12). The interesting morphogenesis of the melanosomelike granules in the present study has an unusual deposition of internal microtubules in certain stages. These microtubules become even more striking when they are viewed in cross sections and are seen to contain central elements which measure approximately 50 A in diameter. To the knowledge of the author this is the first time anyone has reported 50 A central elements inside of microtubules. Thus it is intriguing to speculate that the microtubules and their central elements may be associated with the transport of metabolites during granular development. The release of melanosomelike granules can only be conjectured. However on occasion the whole granular hemocyte heavily laden with dense granules appears to be disintegrating and is very suggestive of a possible mechanism for freeing the granules since the surrounding cells seem intact and well preserved. The plastid-like structures seem to be distinct inclusions and appear not to be a
UNIQUE STRUCTURES IN INSECT HEMOCYTES
657
developmental stage of anything else. Since the mitochondria are small and few in number and since there are no glycogen granules present, it is conceivable although highly speculative that these membrane-containing components may be implicated as an energy source for the circulating granular hemocytes. At this time however their function remains obscure. Recently Locke and McMahon (13) have reported microbodies in the fat body, oenocytes, and Malpighian tubules of an insect. The present study now suggests that microbodies are also present in hemocytes. Peroxidases have been found in insect hemocytes by Ermin (5) and Ohuye (16). de Duve (4) suggested that the catalase of microbodies acts mainly as a peroxidase. Probably these enzymes are located in the microbody-like granules of the granular hemocytes. Marginal bands of microtubules in vertebrates have been reported in erythrocytes of fish, amphibians, reptiles, and birds (6, 7, 14). Also marginal bands have been described in thrombocytes of fish (7), hamsters (8), and cattle (20). In the invertebrates a similar microtubule orientation has been reported recently in the granular hemocytes of an insect (1). The present study of insect granular hemocytes appears to have a comparable marginal band of microtubules. This micro'tubular system without the micro-microtubules is suggestive of a cytoskeletal function (7) and may be primarily concerned with the cell shape. That the granular hemocyte has unusual structures is evident in this study. The variety of internal configurations found in the specific granules in this cell might indicate a more fundamental role than previously stated. These inclusions however should be considered in future studies and should have heuristic value. The author is grateful to Mrs Maria Demeri for her invaluable technical assistance, to Mr Alfred E. Revzin for maintenance of the electron microscope, and to Mr Osmay Yalis for photographic assistance. This work was supported by Grant HE-12741 of the U.S. Public HealtIr Service.
REFERENCES 1. BAERWALD, R. J. and BousH, G. M., J. Ultrastruct. Res. 31, 151 (1970). 2. BIRBECK, M. S. C., Ann. N.Y. Acad. Sci. 100, 540 (1963). 3. 4. 5. 6. 7. 8. 9. 10.
BISALPUTRA,T. and BISALPUTRA,A. A., J. Ultrastruct. Res. 32, 417 (1970). DE DUVE, C., J. Cell Biol. 27, 25A (1965). ERMIN, R., Z. Zellforsch. Mikrosk. Anat. 71, 41 (1939). FAWCETT,D. D., Circulation. 26, t105 (1962). FAWCETT,D. W. and WITEBSKY,F., Z. Zellforsch. Mikrosk. Anat. 62, 785 (1964). HAYDON,G. B. and TAYLOR,D. A., J. Cell Biol. 26, 673 (1965). JONES,J. C., J. Morphol. 99, 233 (1956). -Amer. Zool. 2, 209 0962).
658
HAGOPIAN
11. KAPLAN, M. L., Bull. Brooklyn Entomol. Soe. 51, 1 (1956). 12. KAWASE,S., Y. Insect Physiol. 5, 335 (1960). 13. LOCKE, M. and McMAHoN, J. T., Proe. 27th Annu. E M S A Meeting, p. 148. Claitor's, Baton Rouge, Louisiana, 1970. 14. MASER, M. D. and PmLPOTT, C. W., Anat. Rec. 150, 365 (1964). 15. MAUL, G. G., J. Ultrastruet. Res. 26, 163 (1969). 16. OHUYE, T., Mem. Ehime Univ. (Sci.). 1, 21 (1950). 17. RIZKI, M. T. M. and RIZKI, R. M., J. Biophys. Bioehem. Cytol. 5, 235 (1959). 18. SALT, G., Proc. Roy. Soc. Ser. B 147, 167 (1957). 19. SEUl, M., BmBECK, M. S. C. and FITZPATRICK, T. B., Ann. N.Y. Acad. Sei. 100, 497 (1963). 20. SIMPSON,C. F., Cornell Vet. 57, 390 (1967). 21. VERCAUTEREN,R. E. and AERTS, F., Enzymology 20, 167 (1958).
646
j. ULTRASTRUCTURERESEARCH36, 646--658 (197l)
Unique Structures in the Insect Gronulor Hemocytes MARTIN HAGOPIAN Delzartment of Pathology, New York Medical College, New York, New York 10029 Received November 23, 1970, and in revised form February 4, 1971 The ultrastructural studies of the cockroach nymph granular hemocytes show structures in the cytoplasm that have never been described. Some of the inclusions have structural characteristics suggestive of melanosomes and premelanosomes, plastids, and microbodies. It is particularly interesting that some granules contain microtubules (250 A in diameter) which display central elements. The central elements are approximately 50 A in diameter and reveal 8 spokes radiating from them. In addition the granular hemocytes show marginal bundles of microtubules which are always in close proximity to the cell membrane. These tubules lack the 50 it central elements. The findings are discussed in terms of previous known functions and histochemistry of insect hemocytes. Some of the known functions of insect hemocytes are phagocytosis, encapsulation, melanization, wound healing, coagulation, and possible transformation into other tissues. Also hemocytes have been reported to lyse among differentiating tissues and appear to have a nutritive effect [see Jones (10) for review]. There are several different types of hemocytes, but little is known about their fine structure. Previous studies of these cells lack the ultrastructural morphology that is helpful in determining cellular function. Therefore it would be significant to further elucidate the role of hemocytes by such morphological studies. The purpose of the present study is to describe some cytoplasmic structures and the various stages of development of the highly ordered granules in the cockroach nymph granular hemocytes. The findings are discussed in terms of previous known functions and histochemistry of insect hemocytes.
MATERIALS AND METHODS Femoral segments from the metathoracic leg (hind leg) of the freshly molted cockroach nymph, Leucophaea maderae, were removed and placed into phosphate-buffered 5 % glutaraldehyde at pH 7.4 for 3 hours (room temperature). While in the fixative the exoskeleton was perforated to ensure proper fixation. The tissue was then rinsed in 0.2 M phosphate buffer for 15 hours and postfixed in 2% osmium tetroxide (phosphate-buffered at pH 7.4) for 1 hour at room temperature. Dehydration in various grades of acetone and embedding in Araldite followed. Thin sections were doubly stained with uranyl acetate and lead citrate and were examined in a Philips EM 200 or a Siemens 1A electron microscope.
UNIQUE STRUCTURESIN INSECT HEMOCYTES
647
RESULTS The femoral cuticle with the underlying epidermal cells of the cockroach nymph enclosed the extensor and flexor muscles, nerves, tracheae, tracheoles, and the hemocoel which contains hemolymph and hemocytes. In this open circulatory system the granular hemocytes (perhaps several types) in the hemolymph are conspicuous by their general elliptical appearance, large nucleus, and prominent cytoplasmic granules (Fig. 1). The cytoplasm also contains mitochondria (small and few in number), extensive Golgi regions, rough and smooth endoplasmic reticulum, free ribosomes, lysosome-like bodies, and marginal bundles of microtubules. Glycogen granules appeared to be absent. Although evidence of mitosis has never been noted, these circulating blood cells are present in considerable numbers in the freshly molted n y m p h (within an hour after ecdysis).
Melanosome-like and premelanosome-like granules The most abundant type of granules are the membrane-bounded, roughly spherical, opaque inclusions which average about 1-2 # in diameter (Fig. 2). No inner structure of these inclusions may be resolved even at high magnifications. These dense inclusions resemble vertebrate melanosomes. Other granules have a highly organized substructure (Figs. 3-7) and are reminiscent of the vertebrate premelanosomes (2, 15, 19). Figure 3 shows that the earliest stage of the premelanosome-like inclusions seems to be derived from the Golgi region whereas Figs. 4-7 demonstrate the deposition of membranes inside these granules at various stages of morphogenesis. In Fig. 8 the internal configurations show their concentric arrangement. Furthermore, certain planes of sections of these granules reveal that the internal configurations are microtubules which measure about 250 A in diameter (Figs. 9 and 10). Of particular interest (at a high magnification and at the proper orientation) are the centers of the microtubules. The centers suggest micro-microtubules even though no evidence is presented concerning the three-dimensional structure of the central element. These small tubules are approximately 50 A in diameter and reveal 8 spokes radiating from them (Fig. 11, arrowhead). Perhaps because the central element is so small, it is demonstrated in very FIO. 1. An electron micrograph of a granular hemocyte. It is characterized by its elliptical shape, large nucleus, and prominent dense granules, x 20 000. FIG. 2. A micrograph of a melanin-like granule. It is membrane-bounded and has a dense unstructured matrix, x 40 000. FIG. 3. Portion of a granular hemocyte showing the possible derivation of the early premelanosomelike granules (P) from the Golgi region (G). MG, melanin-like granule; MT, microtubules, x 30 000. FIo. 4. An early premelanosome-like granule displaying the deposition of some internal encircling membranes (arrowhead). x 50 000. FIG. 5. View of a premelanosome-like body demonstrating the internal membrane arrangement. This symmetrical orientation resembles the vertebrate premelanosome, x 40 000.
~i~,~,i~i,~,,,,i,~,i~?i~i~i!,!~iil
"i!,,!,ii,i~i,i!,~!~ii!i!i~i i,!~'i
~
~~ ' 7 ~ ~
~~ % ~ ! ~
~'~!~
~~ 7 ~
650
HAGOPIAN
few cases. Also it is noted in Fig. 11 that if the orientation is not exactly right neither the micro-microtubules nor the spokes radiating f r o m them are visible. In the same micrograph the outer m e m b r a n e defining the premelanosome-like granule exhibits a typical unit membrane. At the different stages of granular morphogenesis the density of the premelanosome-like granules increase until they ultimately become opaque and unstructured. On occasion the whole granular hemocyte laden with dense granules appears to be disintegrating. However the possibility of fixation damage can not be ignored even t h o u g h surrounding cells seem well preserved.
The plastid-like structures These elongated structures measure a b o u t 1.0-1.5 # in length and are approximately 0.2-0.4 # in width. A l t h o u g h they are b o u n d e d by a single membrane, their overall appearance resembles chloroplasts of lower plant forms which are always defined by a double m e m b r a n e (3). Within, the internal membranes generally extend f r o m one end of the long axis to the other end in parallel arrays (Figs. 12 and 13). Furthermore the membranes vary in n u m b e r and often show a localized dense spot at one end (Fig. 12).
Microbody-like granules The microbody-like granules are often encountered in the granular hemocytes. These granules are b o u n d e d by a unit membrane, are spherical in shape, and measure 0.5-1.0 # in diameter (Fig. 14). The finely granular matrix is fairly h o m o g e n e o u s and has a moderate electron opacity. Because of the finer granular matrix, these bodies are distinguishable f r o m early stages of premelanosome-like granules. N o crystalline inclusions have ever been observed in the matrices of these bodies.
Marginal bundles of rnicrotubules Marginal bands, consisting of bundles of microtubules, are seen in the granular hemocytes. The microtubules are arranged in loosely assembled bundles and are Fro. 6. A group of premelanosome-like granules at various orientations. × 38 000. F:G. 7. A more advanced stage of premelanosome-like granules than that in Fig. 6. The granules have more internal membranes and are denser, x 31 000. FIG. 8. Another plane of section of the premelanosome-like granules showing the concentric arrangement of the internal membranes. × 38 000. FIG. 9. At a higher magnification and at the proper orientation the internal configuration may be seen to be microtubules which measure approximately 250/~ in diameter, x 63 000. F:G. 10. Another view of a premelanosome-like granule illustrating the internal microtubules. x 54 000. FIG. 11. A high-power micrograph of a premelanosome-like granule. Especially note the central elements, which measure about 50 ~ in diameter, in the center of the microtubules. These small tubules have 8 spokes radiating from them (arrowhead). The microtubules and central elements may function as a passageway for the transport of metabolites in the granule. Also note the unit membrane limiting the granule (1). x 240 000.
UNIQUE STRUCTURES IN INSECT HEMOCYTES
651
> Z
©
>
UNIQUE STRUCTURES IN INSECT HEMOCYTES
653
654
HAGOPIAN
FIGS. 12 and 13. Examples of plastid-like structures (PS). These membrane-containing components are bounded by a single membrane. The internal membranes, which vary in number, generally extend from one end of the long axis to the other end in parallel arrays, and occasionally a dense spot (arrowhead) is found at one end. The function of these inclusions remains obscure. MT, microtubules. Fig. 12, × 40 000. Fig. 13, x 42 000.
UNIQUE STRUCTURES IN INSECT HEMOCYTES
655
FIG. 14. A microbody-like inclusion. These unit membrane-bounded granules are spherical in shape and measure 0.5-1.0/~ in diameter. The finely granular matrix has a moderate electron opacity. x 72 000. Fro, 15. Marginal bundles of microtubules about 250/~ in diameter (MT) are loosely assembled in close proximity to the granular hemocyte plasma membrane. In cross sections these microtubules do not have the centrally located 50/~ micro-microtubules that are found in some of the premelanosomelike granules. The marginal tubules appear to encircle the cell and are suggestive of a cytoskeletal function. H, precipitated hemolymph, x 50 000.
656
HAGOPIAN
always in close proximity to the cell membrane. The tubules are never connected to the surface of the cell or to each other and seem to encircle the disk-shaped cell. In certain planes of section they manifest themselves in cross or slightly tangential orientation at the poles of the cells (Figs. 3 and 13). Other planes of section reveal marginal arrangements of longitudinally oriented microtubules (Fig. 15). In cross sections the tubules vary from 25 to 35 per bundle, are surrounded by a halo, and are about 250 A in diameter. These microtubules lack the centrally located 50 A micro-microtubules that are seen in the microtubules of some granules. DISCUSSION The granular hemocytes of the cockroach nymph reveal well-preserved structures in their cytoplasm that have never been described. Many of the highly organized granules in the multifunctional, circulating blood cells appear to be in various stages of morphogenesis. At times some of these developing granules and some of the other inclusions show structural characteristics suggestive of melanosomes and premelanosomes, plastids, and microbodies (peroxisomes). Such findings add supportive morphological evidence to known functions and histochemistry of hemocytes [see Jones (10) for review] and also present additional information. The most common and most striking inclusions are those that resemble the melanosomes and premelanosomes found in vertebrate melanocytes and melanomas (2, 15, 19). In support of this resemblance Kaplan (11) has reported that in some specific cases hemocytes of insects may be involved in the melanization of encapsulated material. Salt (18) has shown that hemocytes in the developmental stages of Tenebrio envelop and melanize eggs of an ichneumon fly. Also tyrosinase has been found as a chemical constituent of hemocytes by Jones (9), Vercauteren and Aerts (21), Rizki and Rizki (17), and Kawase (12). The interesting morphogenesis of the melanosomelike granules in the present study has an unusual deposition of internal microtubules in certain stages. These microtubules become even more striking when they are viewed in cross sections and are seen to contain central elements which measure approximately 50 A in diameter. To the knowledge of the author this is the first time anyone has reported 50 A central elements inside of microtubules. Thus it is intriguing to speculate that the microtubules and their central elements may be associated with the transport of metabolites during granular development. The release of melanosomelike granules can only be conjectured. However on occasion the whole granular hemocyte heavily laden with dense granules appears to be disintegrating and is very suggestive of a possible mechanism for freeing the granules since the surrounding cells seem intact and well preserved. The plastid-like structures seem to be distinct inclusions and appear not to be a
UNIQUE STRUCTURES IN INSECT HEMOCYTES
657
developmental stage of anything else. Since the mitochondria are small and few in number and since there are no glycogen granules present, it is conceivable although highly speculative that these membrane-containing components may be implicated as an energy source for the circulating granular hemocytes. At this time however their function remains obscure. Recently Locke and McMahon (13) have reported microbodies in the fat body, oenocytes, and Malpighian tubules of an insect. The present study now suggests that microbodies are also present in hemocytes. Peroxidases have been found in insect hemocytes by Ermin (5) and Ohuye (16). de Duve (4) suggested that the catalase of microbodies acts mainly as a peroxidase. Probably these enzymes are located in the microbody-like granules of the granular hemocytes. Marginal bands of microtubules in vertebrates have been reported in erythrocytes of fish, amphibians, reptiles, and birds (6, 7, 14). Also marginal bands have been described in thrombocytes of fish (7), hamsters (8), and cattle (20). In the invertebrates a similar microtubule orientation has been reported recently in the granular hemocytes of an insect (1). The present study of insect granular hemocytes appears to have a comparable marginal band of microtubules. This micro'tubular system without the micro-microtubules is suggestive of a cytoskeletal function (7) and may be primarily concerned with the cell shape. That the granular hemocyte has unusual structures is evident in this study. The variety of internal configurations found in the specific granules in this cell might indicate a more fundamental role than previously stated. These inclusions however should be considered in future studies and should have heuristic value. The author is grateful to Mrs Maria Demeri for her invaluable technical assistance, to Mr Alfred E. Revzin for maintenance of the electron microscope, and to Mr Osmay Yalis for photographic assistance. This work was supported by Grant HE-12741 of the U.S. Public HealtIr Service.
REFERENCES 1. BAERWALD, R. J. and BousH, G. M., J. Ultrastruct. Res. 31, 151 (1970). 2. BIRBECK, M. S. C., Ann. N.Y. Acad. Sci. 100, 540 (1963). 3. 4. 5. 6. 7. 8. 9. 10.
BISALPUTRA,T. and BISALPUTRA,A. A., J. Ultrastruct. Res. 32, 417 (1970). DE DUVE, C., J. Cell Biol. 27, 25A (1965). ERMIN, R., Z. Zellforsch. Mikrosk. Anat. 71, 41 (1939). FAWCETT,D. D., Circulation. 26, t105 (1962). FAWCETT,D. W. and WITEBSKY,F., Z. Zellforsch. Mikrosk. Anat. 62, 785 (1964). HAYDON,G. B. and TAYLOR,D. A., J. Cell Biol. 26, 673 (1965). JONES,J. C., J. Morphol. 99, 233 (1956). -Amer. Zool. 2, 209 0962).
658
HAGOPIAN
11. KAPLAN, M. L., Bull. Brooklyn Entomol. Soe. 51, 1 (1956). 12. KAWASE,S., Y. Insect Physiol. 5, 335 (1960). 13. LOCKE, M. and McMAHoN, J. T., Proe. 27th Annu. E M S A Meeting, p. 148. Claitor's, Baton Rouge, Louisiana, 1970. 14. MASER, M. D. and PmLPOTT, C. W., Anat. Rec. 150, 365 (1964). 15. MAUL, G. G., J. Ultrastruet. Res. 26, 163 (1969). 16. OHUYE, T., Mem. Ehime Univ. (Sci.). 1, 21 (1950). 17. RIZKI, M. T. M. and RIZKI, R. M., J. Biophys. Bioehem. Cytol. 5, 235 (1959). 18. SALT, G., Proc. Roy. Soc. Ser. B 147, 167 (1957). 19. SEUl, M., BmBECK, M. S. C. and FITZPATRICK, T. B., Ann. N.Y. Acad. Sei. 100, 497 (1963). 20. SIMPSON,C. F., Cornell Vet. 57, 390 (1967). 21. VERCAUTEREN,R. E. and AERTS, F., Enzymology 20, 167 (1958).