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Bombyx mori prohemocyte division and differentiation in individual microcultures
Journal of Insect Physiology 47 (2001) 325–331 www.elsevier.com/locate/jinsphys
Bombyx mori prohemocyte division and differentiation in individual microcultures Miyuki Yamashita 1, Kikuo Iwabuchi
*
Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu Tokyo, 183-8509 Japan Received 4 January 2000; accepted 16 October 2000
Abstract We followed the fate of microcultured Bombyx mori prohemocytes in vitro. Prohemocytes isolated from larval hemolymph (day 1 of 4th instar) were maintained for 4–11 days in serum-free MGM-450 medium and some of them underwent mitotic division. Over 60% of the non-dividing prohemocytes differentiated to plasmatocytes or granulocytes. Some of the granulocytes subsequently transformed to spherulocytes. Of the dividing prohemocytes, 59.2% of the daughter cells differentiated into other types of hemocytes such as plasmatocytes, granulocytes and spherulocytes, and the remainder divided into new prohemocytes. Four of these renewed prohemocytes generated daughter cells composed of plasmatocytes and granulocytes. These results suggest that prohemocytes possess the properties of stem cells, and that plasmatocytes and spherulocytes may be terminally differentiated cells, whereas granulocytes, at least in part, may be a transient form of spherulocyte. Oenocytoids were not produced, suggesting that the lineage of oenocytoids differs from that of other types of hemocytes and that it is determined before release from hemopoietic organs. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bombyx mori; Hemocytes; Differentiation; Culture
1. Introduction Insect hemocytes consist of a complex of several types of mesodermal cells that circulate in the blood. Nittono (1964) identified five types of B. mori hemocytes: proleucocytes (designated prohemocytes in this study), plasmatocytes, granulocytes, spherulocytes, and oenocytoids. These represent fairly well-defined groups that are morphologically distinct and have broad functionality within Lepidoptera. These cells are thought to be supplied by hemopoiesis in the hemopoietic organs or by mitotic division of circulating hemocytes (Jones, 1970; Akai and Sato, 1971; Arnold and Hinks, 1976). The question of whether the different types of cells represent transient morphological variants of one kind of cell or constitute a number of distinctive and immutable types, remains unanswered. Several investigators disagree over * Corresponding author. Tel.: +81-423-67-5693; fax: +81-423-675693. E-mail address: [email protected] (K. Iwabuchi). 1 Present address: Pharmaceutical Research Laboratories, Taisho Pharmaceutical Co. Ltd, Ohmiya, Saitama, Japan.
the origins of the various types of hemocytes, and it appears that several differentiation pathways exist in various insects (Arnold, 1979). Arnold (1979) categorized the prevailing opinions into single-cell and multiplecell theories. The monophyletic theory of the origin of hemocytes states that one stem cell differentiates into various cell types under appropriate conditions. The polyphyletic theory states that each type of cell is derived from a different stem cell. To prove either of these theories, the differentiation steps from one type of hemocyte to the next must be demonstrated and the conditions of differentiation should be investigated (Feir, 1979). Prohemocytes are located in hemopoietic organs and in hemolymph. These cells are considered to be the stem cells from which the main types differentiate, according to morphological (Gupta and Sutherland, 1966; Beeman et al., 1983), ultrastructural (Shrestha and Gateff, 1982) and radioisotopic (Shrivastava and Richards, 1965; Peake, 1979; Peake and Crossley, 1979; Lea, 1986) studies. If so, prohemocytes should be able to self-renew and to generate differentiated progeny. The mitotic activity of prohemocytes has been consistently reported (Jones,
0022-1910/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 0 ) 0 0 1 4 4 - X
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1962; Arnold and Hinks, 1976; Breugnon and Le Berre, 1976). Shrivastava and Richards (1965) first suggested that Galleria mellonella prohemocytes, granulocytes and plasmatocytes form one lineage, whereas spherulocytes and oenocytoids arise from different lineages. Beaulaton (1979) supposed from observations of intermediate hemocyte types of B. mori, that prohemocytes generate pluripotent plasmatocytes that can produce granulocytes, spherulocytes and oenocytoids. Hoffmann (1967) suggested that Locusta migratoria prohemocytes differentiate into plasmatocytes. Although these studies provided valuable support for the notion of differentiation in prohemocytes, none of them directly followed the fate of prohemocytes. Culture in vitro can reveal much information about the differentiation of hemocytes. Primary culture should be started from individual hemocytes obtained directly from insects to determine whether or not they will produce cells of their own kind and whether or not the progeny will differentiate into other hemocyte types. However, all in vitro studies to date have begun with mixed hemocyte population, which has prevented opportunities to examine the differentiation and fate of individual cells over a long period. Single-cell cultures should resolve this. By following the fate of individual B. mori prohemocytes in culture, we show here that the prohemocytes circulating in the blood can renew themselves and generate various types of progeny.
2. Materials and methods 2.1. Insects Larvae of the silkworm, B. mori (F1 hybrids between two inbred varieties: J122×C115), reared on an artificial diet (Nihon Nosan Kogyo, Yokohama) at 25±1°C under a 16:8 (L:D) photoperiod were staged on the day of the 3rd ecdysis. This day was designated as day 1 of the 4th instar (L4D1). 2.2. Preparation and culture of prohemocytes L4D1 larvae were chilled in crushed ice for 15 min, immersed in 70% ethanol for 1 min. Thereafter, 1.0–1.5 ml of cold anticoagulant (0.098 M NaOH, 0.146 M NaCl, 0.017 M EDTA (free acid) and 0.041 M citric acid (pH 4.5 and osmolality 370 Osm kg⫺1)) (Mead et al., 1986) was injected via a 27 G needle inserted into the hemocoel through a proleg. The larvae were then kept at room temperature for 1–2 min. Hemolymph obtained by cutting a proleg was collected into 35 mm plastic dishes containing 2 ml of cold anticoagulant. The hemolymph/anticoagulant mixture was then transferred into a 10 ml centrifuge tube and cells were sedimented by centrifugation at 100g for 5 min. The cells were
washed twice with culture medium and then resuspended in 2 ml of culture medium. The cell suspension was transferred to a Petri dish. Individual prohemocytes were isolated from the suspension in 10 µl culture medium using a hand-pulled glass microelectrode and transferred to the base of Terasaki wells at a density of one cell per well. All cultures were incubated at 25°C, then observed by phase microscopy 1–2 h after plating to confirm that each well contained a single prohemocyte. The fate of the cells was followed by daily observation until the cells died. 2.3. Culture media The basic medium was MGM-450 (Mitsuhashi and Inoue, 1988) without fetal bovine serum (FBS). Various media supplemented with B. mori hemolymph were also prepared. Hemolymph was collected from L5D2, L5D4, and L5D6 B. mori larvae surface-sterilized with 70% ethanol in a cold vial. The hemolymph was heated at 60°C for 10 min to prevent melanization, then stored at ⫺40°C. The hemolymph was thawed, and clarified by centrifugation for 15 min at 700g before use. The supernatant was diluted in 10 or 50% using the basic medium. 2.4. Morphological identification of hemocytes Hemocytes were classified according to Nittono (1964), but the terminology follows that of Wago (1991). Briefly, B. mori hemocytes were classified according to morphological criteria into prohemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. Prohemocytes are small, round non-adhesive cells with a prominent nucleus and thin cytoplasm without cytoplasmic inclusions [Fig. 1(a)]. These cells occupy a small portion of the hemocyte population in hemolymph (3.3% in L4D1 B. mori larvae). Granulocytes are round or ovoid with numerous small granules, but when seeded on culture plates, they adhere to the surface of the plates and spread uniformly with each axis of the cell being approximately equal in length. These responses usually occurred within 10 min in vitro after withdrawal from the hemocoel. However, the granulocytes obtained using the anticoagulant techniques mentioned above took at least 20 min to adhere and 40 min to spread fully in vitro [Fig. 1(d) and (e)]. Plasmatocytes are oval or spindle-shaped, but highly pleomorphic, without cytoplasmic inclusions. These cells are also adherent and usually spread asymmetrically with one axis of the cell almost always longer than the other. These plasmatocyte responses usually occurred within 10 min after placing the cells in vitro. When obtained using the anticoagulant techniques, the structure of the plasmatocytes was oval with a few small protrusions, but did not adhere to the substratum. The cells took at least 2 h to protrude lamellopodia on the substratum and 3 h to spread fully in vitro
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larval hemolymph, five cells divided twice and survived for an average of 16.1 days (range: 7–22 days). 3.2. Differentiation of prohemocytes in vitro
Fig. 1. Morphology of different types of hemocytes first placed into culture. (a) Prohemocyte, (b) spherulocyte, (c) oenocytoid, unlysed, (d) granulocyte, after 10 min of culture, (e) granulocyte, which partially protruded filopodia, after 20 min of culture, (f) fully spread granulocyte, after 40 min of culture, (g) plasmatocyte, after 10 min of culture, (h) early-spreading plasmatocyte, after 2 h of culture, and (i) fully spread plasmatocyte, after 3 h of culture. Scale bar=10 µm.
[Fig. 1(g), (h) and (i)]. Spherulocytes and oenocytoids are non-adhesive [Fig. 1(b) and (c)]. Spherulocytes contain large, cytoplasmic inclusions, whereas oenocytoids are large pleomorphic cells with homogeneous cytoplasm that is devoid of cytoplasmic inclusions. The B. mori hemocytes were morphologically distinct enough for identification under a phase-contrast microscope.
3. Results 3.1. Survival duration and mitotic division of prohemocytes in vitro Over 94% of the prohemocytes individually cultured in the basic medium survived the first 24 h of culture, but none of them underwent division. The survival rate and ratio of dividing cells were improved by adding larval hemolymph to the medium. When the medium contained 10% larval hemolymph, 8.1% of the cells underwent mitotic division during the first 24 h of culture (Fig. 2). At 50% hemolymph, this ratio increased to 10.7%. The average survival of dividing prohemocytes was 9.7–12.6 days, which was longer than that of non-dividing cells. By the end of culture, approximately 20% of prohemocytes that were incubated in medium containing hemolymph underwent mitotic division, compared with 1.8% in basic medium alone (Table 1). Among the prohemocytes cultured in medium containing
We examined the morphological changes undergone by cultured prohemocytes. Thirty two percent of nondividing prohemocytes cultured in basic medium differentiated to granulocytes (Table 2). The differentiation occurred 1–8 d of culture (3.5 d on average). The first phase of differentiation was an appearance of small granules in cytoplasm, giving rise to a round-shaped granulocyte containing many prominent granules, identical to the granulocyte just collected from the hemocoel [Fig. 3(a)]. The granulocytes extended filopodia 1–3 d after differentiation [Fig. 3(b)], and fully spread to adhere to the substratum 3–5 d after differentiation [Fig. 3(c)]. Thirty five percent of non-dividing prohemocytes differentiated to plasmatocytes (Table 2). The differentiation occurred following 1–7 d of culture (2.5 d on average). These cells first became spindle-shaped or oval cells without cytoplasmic inclusions, identical to the plasmatocytes just collected from the hemocoel [Fig. 3(e)]. The plasmatocytes adhered to the substratum 1–2 d after differentiation [Fig. 3(f)], and then became amoeboid with lamellopodia which are exclusive features of plasmatocytes 2–3 d after differentiation [Fig. 3(g)]. Eventually 67.9% of the prohemocytes differentiated and the remainder did not. The differentiated cells survived for over 2 days after differentiation. These results indicate that B. mori prohemocytes circulating in the blood can generate differentiated progeny such as plasmatocytes and granulocytes. Among the prohemocytes that mitotically divided in medium containing larval hemolymph, 59.2% of daughter cells became other types of hemocytes such as plasmatocytes, granulocytes and spherulocytes, whereas the remaining 40.8% divided into new prohemocytes (Table 3). Approximately 55% of the differentiation occurred 2–11 d after mitotic division, whereas 45% occurred within 24 h after division. Morphological changes of the differentiated hemocytes and the rate were essentially the same as those of the hemocytes differentiated from non-dividing prohemocytes. The composition of differentiated daughter cells indicated that 55.3% of cell divisions were symmetrical (single-type) and the remainder were asymmetrical (mixed-type). Among the asymmetrically divided prohemocytes, four cells gave rise to daughter cells composed of plasmatocytes and granulocytes, suggesting that these types of hemocytes form cells of the same lineage. These results showed that each prohemocyte can differentiate into different types of hemocytes, suggesting that the fate of B. mori prohemocytes is not predestined, even after dispersion from hematopoietic organs. Forty percent of prohemocytes were renewed by a symmetrical or asym-
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Fig. 2. Mitotic division of isolated prohemocyte in vitro. (a) Isolated prohemocyte, (b) mitotic phase of the prohemocyte, after 1 d of culture, and (c) two daughter prohemocytes, which separated within 1 h after mitotic phase. Scale bar=10 µm. Table 1 Division and survival of prohemocytes (PR) cultured in MGM-450 medium containing various concentrations of hemolymph ConcentrationTotal no. of % Divisiona Survival of hemocytesa of PR cultured (days) hemolymph (%)
0 10 50
92 99 74
1.8±1.8 19.6±0.8b 22.1±1.7b
Nondividing
Dividing
4.2±1.1 5.3±1.0 6.8±1.4
11.0 9.7±3.8 12.6±2.5c
from L5D2, L5D4 and L5D6 larvae supported 68.5, 60.2 and 28.3% differentiation, respectively (Fig. 4). Under each of these conditions, plasmatocytes and granulocytes were evident as differentiated types of hemocytes. When L5D2 or L5D4 larval hemolymph was added to the medium, the fate of differentiated prohemocytes was granulocyte-biased or non-biased, respectively. 4. Discussion
Values are means of three replicates, ±SD. Significantly higher than value for 0% hemolymph, P⬍0.05, ANOVA. c Significantly longer than period for non-dividing hemocytes, P⬍0.05, ANOVA. a
b
Optical and electron microscopic studies have provided valuable insight into the process of hemocyte differentiation. Devauchelle (1971) observed many intermediate stages among Melolontha melolontha prohemocytes, plasmatocytes, granulocytes and adipohemocytes, suggesting that each type is derived from the other. An ultrastructual and cytological study by Beaulaton and Monpeyssin (1976) suggested that pro-
Table 2 Frequency of cell types (%) differentiated from non-dividing prohemocytes cultured in medium containing various concentrations of hemolympha Concentration of hemolymph (%)
Total no. of PR cultured
PR (non-differentiated) PL (%) (%)
GR (%)
Total % of differentiated PR
0
92 79
50
57
32.5±0.4 (2.7±0.6) 26.1±4.8 (3.5±1.8) 19.6±0.7 (5.5±3.1)
67.9±0.5
10
32.1±0.3 — 40.8±3.1 — 49.8±1.7 —
a b
35.4±1.5 (2.2±0.2)b 33.2±0.1 (2.6±0.7) 26.8±2.3 (2.4±1.0)
59.2±3.1 46.4±1.9
Values are means of three replicates, ±SD. PR, prohemocyte; PL, plasmatocyte; GR, granulocyte. Values in parentheses are survival of differentiated cells (days).
metrical type of mitotic division. Conversely, plasmatocytes and granulocytes that differentiated from prohemocytes in vitro and which did not yet extend filopodia or protrusion, symmetrically divided to generate only the same types of hemocytes (Table 3). 3.3. Effect of hemolymph obtained from different stages of larvae on prohemocyte differentiation in vitro Larval hemolymph supported the differentiation of prohemocytes in vitro. However, hemolymph samples
hemocytes are stem cells that become plasmatocytes after several intermediate stages. Akai and Sato (1971) indicated that several types of differentiated B. mori hemocytes are released from hemopoietic organs through openings in the surrounding acellular membrane. Han et al. (1998) examined hemocytic differentiation in the hemopoietic organs of B. mori larvae, using transmission electron microscopy. Their study indicated that immature and mature hemocytes co-exist. They suggested that the lineage of each type of hemocyte involving prohemocytes in hemolymph can be formed from pluripotent stem cells located in compact islets of hemo-
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Fig. 3. Morphology of different types of hemocytes differentiated from isolated prohemocytes in vitro. (a) Non-adherent granulocyte, after 2 d of culture, (b) granulocyte, which protruded filopodia, 1 d after differentiation, (c) fully spread granulocyte, 2 d after differentiation, (d) spherulocyte, (e) non-adherent oval plasmatocyte, after 2 d of culture, (f) plasmatocyte, 1 d after differentiation, and (g) fully spread plasmatocyte, 2 d after differentiation. Scale bar=10 µm. Table 3 Composition of daughter cells from hemocytes cultured in medium containing 10 or 50% hemolymph Cell type at divisiona
Total no. of dividing hemocytes
Composition of daughter cells
(Single-type)
PR
38
(Mixed-type)
PR PR
PL PL
GR GR
SP SP
PR PL
PR GR
9
2
9
1
7
6
2.3c (1.2)d GRb
5
PLb
2
a b c d e
7.6 (3.6) 5e 5.8
7.0 (1.0)
3.3 (2.4)
3.2 (5.0)
PL GR 4 PL 6.8 (1.8)
GR 5.0 (6.8)
2 7.3
PR, prohemocyte; PL, plasmatocyte; GR, granulocyte; SP, spherulocyte. Hemocytes differentiated from prohemocytes in culture. Values below number of pairs of dividing hemocytes are survival of differentiated cells (days). Values in parenthesis are days from mitotic division to differentiation. Two of these subsequently transformed into spherulocytes before protrusion of filopodia.
poietic organs, and that the formed hemocyte may not undergo further transformation. These studies have provided significant information regarding hemopoiesis, but did not successively follow the fate of specific hemocytes. In the present study, we isolated and cultured individual B. mori prohemocytes from larval hemolymph. When cultured in MGM-450 medium without FBS, 1.8–22.1% of the prohemocytes underwent cell division. Furthermore, many prohemocytes directly obtained from hemolymph or produced by cell division of individually cultured prohemocytes differentiated into granulocytes, plasmatocytes and spherulocytes. Thus, the prohemocytes released from the hemopoietic organs and circulating in the blood are also pluripotent. Gupta and Suther-
land (1966) observed the in vitro transformation of plasmatocytes into various cell types such as granulocytes, spherulocytes and oenocytoids. Mitsuhashi (1966) reported that various cells intermediate between prohemocytes and plasmatocytes appeared in Chilo suppressalis hemocyte cultures, suggesting the transformation of prohemocytes into plasmatocytes. Sohi (1971) also suggested from an in vitro study that plasmatocytes are derived from prohemocytes. In the present study, several prohemocytes divided twice and some of the daughter cells gave rise to granulocytes after the second division (data not shown). Moreover, four prohemocytes produced daughter cells composed of plasmatocytes and granulocytes. These findings demonstrated that B. mori prohemocytes can self-renew and are pluripotent, sug-
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Fig. 4. Types of hemocytes differentiated from prohemocytes cultured in medium containing 10% hemolymph collected from various ages (L5D2, L5D4 and L5D6) of fifth instar B. mori larvae.
differentiation depended upon the stage of larvae from which it was obtained. These findings suggested that specific stimulants and/or materials are required to support hemocyte development in vitro. Many earlier studies of B. mori (e.g., Nittono, 1960) have noted that differential and total hemocyte counts fluctuate with the molting cycle, suggesting that hemopoiesis and the activity of hemocytes are under endocrine control. Hoffmann and Joly (1969) and Hoffmann (1970) found that the corpora allata influences both the differentiation and production of hemocytes in Locusta migratoria. Han et al. (1995) reported that 20-hydroxyecdysone (20OHE) and juvenile hormone, particularly 20-OHE, activates the discharge of hemocytes from hemopoietic organs in B. mori. Thus, the effects of insect hormones on prohemocyte differentiation should be examined.
Acknowledgements gesting that they have features characteristic of stem cells. However, oenocytoids were not differentiated from prohemocytes in the present study, although the former are a type of hemocyte in B. mori. These results suggested that the lineage of oenocytoids differs from that of other types of hemocytes. Thus, this study indicated that various types of hemocytes, with the exception of oenocytoids, can be generated by differentiation of circulating prohemocytes in addition to hemopoiesis in hemopoietic organs. Plasmatocytes and granulocytes predominate in B. mori hemolymph, and are associated with cellular defence against invading pathogens or parasitoid eggs, as well as the disposal of waste cells produced through larval development and metamorphosis. This study showed that these cells also undergo mitotic division in vitro and give rise to their own types of daughter cells. However, some of the granulocytes subsequently transformed into spherulocytes. Cockroach granulocytes differentiate into spherulocytes (Gupta and Sutherland, 1967). These facts indicate that granulocytes are not terminally differentiated. Granulocytes may be heterogeneous with respect to their fate, since the cells can be immunologically grouped into several sub-types (Brehelin and Zachary, 1986). Gardiner and Strand (1999) suggested using monoclonal antibodies against Pseudoplusia includens hemocytes to show that granulocytes are antigenetically related to spherulocytes. The present study showed that plasmatocytes and spherulocytes of B. mori may be terminally defferentiated cells, but granulocytes are not. Further studies are needed to determine whether B. mori granulocytes are heterogeneous with respect to their fate, or whether they are a transient form of spherulocytes. The survival and development of cultured prohemocytes were improved by including larval hemolymph in the medium. The effect of hemolymph on growth and
We are grateful to Dr H. Fugo for providing silkworm stocks.
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Bombyx mori prohemocyte division and differentiation in individual microcultures Miyuki Yamashita 1, Kikuo Iwabuchi
*
Tokyo University of Agriculture and Technology, Saiwai-cho, Fuchu Tokyo, 183-8509 Japan Received 4 January 2000; accepted 16 October 2000
Abstract We followed the fate of microcultured Bombyx mori prohemocytes in vitro. Prohemocytes isolated from larval hemolymph (day 1 of 4th instar) were maintained for 4–11 days in serum-free MGM-450 medium and some of them underwent mitotic division. Over 60% of the non-dividing prohemocytes differentiated to plasmatocytes or granulocytes. Some of the granulocytes subsequently transformed to spherulocytes. Of the dividing prohemocytes, 59.2% of the daughter cells differentiated into other types of hemocytes such as plasmatocytes, granulocytes and spherulocytes, and the remainder divided into new prohemocytes. Four of these renewed prohemocytes generated daughter cells composed of plasmatocytes and granulocytes. These results suggest that prohemocytes possess the properties of stem cells, and that plasmatocytes and spherulocytes may be terminally differentiated cells, whereas granulocytes, at least in part, may be a transient form of spherulocyte. Oenocytoids were not produced, suggesting that the lineage of oenocytoids differs from that of other types of hemocytes and that it is determined before release from hemopoietic organs. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bombyx mori; Hemocytes; Differentiation; Culture
1. Introduction Insect hemocytes consist of a complex of several types of mesodermal cells that circulate in the blood. Nittono (1964) identified five types of B. mori hemocytes: proleucocytes (designated prohemocytes in this study), plasmatocytes, granulocytes, spherulocytes, and oenocytoids. These represent fairly well-defined groups that are morphologically distinct and have broad functionality within Lepidoptera. These cells are thought to be supplied by hemopoiesis in the hemopoietic organs or by mitotic division of circulating hemocytes (Jones, 1970; Akai and Sato, 1971; Arnold and Hinks, 1976). The question of whether the different types of cells represent transient morphological variants of one kind of cell or constitute a number of distinctive and immutable types, remains unanswered. Several investigators disagree over * Corresponding author. Tel.: +81-423-67-5693; fax: +81-423-675693. E-mail address: [email protected] (K. Iwabuchi). 1 Present address: Pharmaceutical Research Laboratories, Taisho Pharmaceutical Co. Ltd, Ohmiya, Saitama, Japan.
the origins of the various types of hemocytes, and it appears that several differentiation pathways exist in various insects (Arnold, 1979). Arnold (1979) categorized the prevailing opinions into single-cell and multiplecell theories. The monophyletic theory of the origin of hemocytes states that one stem cell differentiates into various cell types under appropriate conditions. The polyphyletic theory states that each type of cell is derived from a different stem cell. To prove either of these theories, the differentiation steps from one type of hemocyte to the next must be demonstrated and the conditions of differentiation should be investigated (Feir, 1979). Prohemocytes are located in hemopoietic organs and in hemolymph. These cells are considered to be the stem cells from which the main types differentiate, according to morphological (Gupta and Sutherland, 1966; Beeman et al., 1983), ultrastructural (Shrestha and Gateff, 1982) and radioisotopic (Shrivastava and Richards, 1965; Peake, 1979; Peake and Crossley, 1979; Lea, 1986) studies. If so, prohemocytes should be able to self-renew and to generate differentiated progeny. The mitotic activity of prohemocytes has been consistently reported (Jones,
0022-1910/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 0 ) 0 0 1 4 4 - X
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1962; Arnold and Hinks, 1976; Breugnon and Le Berre, 1976). Shrivastava and Richards (1965) first suggested that Galleria mellonella prohemocytes, granulocytes and plasmatocytes form one lineage, whereas spherulocytes and oenocytoids arise from different lineages. Beaulaton (1979) supposed from observations of intermediate hemocyte types of B. mori, that prohemocytes generate pluripotent plasmatocytes that can produce granulocytes, spherulocytes and oenocytoids. Hoffmann (1967) suggested that Locusta migratoria prohemocytes differentiate into plasmatocytes. Although these studies provided valuable support for the notion of differentiation in prohemocytes, none of them directly followed the fate of prohemocytes. Culture in vitro can reveal much information about the differentiation of hemocytes. Primary culture should be started from individual hemocytes obtained directly from insects to determine whether or not they will produce cells of their own kind and whether or not the progeny will differentiate into other hemocyte types. However, all in vitro studies to date have begun with mixed hemocyte population, which has prevented opportunities to examine the differentiation and fate of individual cells over a long period. Single-cell cultures should resolve this. By following the fate of individual B. mori prohemocytes in culture, we show here that the prohemocytes circulating in the blood can renew themselves and generate various types of progeny.
2. Materials and methods 2.1. Insects Larvae of the silkworm, B. mori (F1 hybrids between two inbred varieties: J122×C115), reared on an artificial diet (Nihon Nosan Kogyo, Yokohama) at 25±1°C under a 16:8 (L:D) photoperiod were staged on the day of the 3rd ecdysis. This day was designated as day 1 of the 4th instar (L4D1). 2.2. Preparation and culture of prohemocytes L4D1 larvae were chilled in crushed ice for 15 min, immersed in 70% ethanol for 1 min. Thereafter, 1.0–1.5 ml of cold anticoagulant (0.098 M NaOH, 0.146 M NaCl, 0.017 M EDTA (free acid) and 0.041 M citric acid (pH 4.5 and osmolality 370 Osm kg⫺1)) (Mead et al., 1986) was injected via a 27 G needle inserted into the hemocoel through a proleg. The larvae were then kept at room temperature for 1–2 min. Hemolymph obtained by cutting a proleg was collected into 35 mm plastic dishes containing 2 ml of cold anticoagulant. The hemolymph/anticoagulant mixture was then transferred into a 10 ml centrifuge tube and cells were sedimented by centrifugation at 100g for 5 min. The cells were
washed twice with culture medium and then resuspended in 2 ml of culture medium. The cell suspension was transferred to a Petri dish. Individual prohemocytes were isolated from the suspension in 10 µl culture medium using a hand-pulled glass microelectrode and transferred to the base of Terasaki wells at a density of one cell per well. All cultures were incubated at 25°C, then observed by phase microscopy 1–2 h after plating to confirm that each well contained a single prohemocyte. The fate of the cells was followed by daily observation until the cells died. 2.3. Culture media The basic medium was MGM-450 (Mitsuhashi and Inoue, 1988) without fetal bovine serum (FBS). Various media supplemented with B. mori hemolymph were also prepared. Hemolymph was collected from L5D2, L5D4, and L5D6 B. mori larvae surface-sterilized with 70% ethanol in a cold vial. The hemolymph was heated at 60°C for 10 min to prevent melanization, then stored at ⫺40°C. The hemolymph was thawed, and clarified by centrifugation for 15 min at 700g before use. The supernatant was diluted in 10 or 50% using the basic medium. 2.4. Morphological identification of hemocytes Hemocytes were classified according to Nittono (1964), but the terminology follows that of Wago (1991). Briefly, B. mori hemocytes were classified according to morphological criteria into prohemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. Prohemocytes are small, round non-adhesive cells with a prominent nucleus and thin cytoplasm without cytoplasmic inclusions [Fig. 1(a)]. These cells occupy a small portion of the hemocyte population in hemolymph (3.3% in L4D1 B. mori larvae). Granulocytes are round or ovoid with numerous small granules, but when seeded on culture plates, they adhere to the surface of the plates and spread uniformly with each axis of the cell being approximately equal in length. These responses usually occurred within 10 min in vitro after withdrawal from the hemocoel. However, the granulocytes obtained using the anticoagulant techniques mentioned above took at least 20 min to adhere and 40 min to spread fully in vitro [Fig. 1(d) and (e)]. Plasmatocytes are oval or spindle-shaped, but highly pleomorphic, without cytoplasmic inclusions. These cells are also adherent and usually spread asymmetrically with one axis of the cell almost always longer than the other. These plasmatocyte responses usually occurred within 10 min after placing the cells in vitro. When obtained using the anticoagulant techniques, the structure of the plasmatocytes was oval with a few small protrusions, but did not adhere to the substratum. The cells took at least 2 h to protrude lamellopodia on the substratum and 3 h to spread fully in vitro
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larval hemolymph, five cells divided twice and survived for an average of 16.1 days (range: 7–22 days). 3.2. Differentiation of prohemocytes in vitro
Fig. 1. Morphology of different types of hemocytes first placed into culture. (a) Prohemocyte, (b) spherulocyte, (c) oenocytoid, unlysed, (d) granulocyte, after 10 min of culture, (e) granulocyte, which partially protruded filopodia, after 20 min of culture, (f) fully spread granulocyte, after 40 min of culture, (g) plasmatocyte, after 10 min of culture, (h) early-spreading plasmatocyte, after 2 h of culture, and (i) fully spread plasmatocyte, after 3 h of culture. Scale bar=10 µm.
[Fig. 1(g), (h) and (i)]. Spherulocytes and oenocytoids are non-adhesive [Fig. 1(b) and (c)]. Spherulocytes contain large, cytoplasmic inclusions, whereas oenocytoids are large pleomorphic cells with homogeneous cytoplasm that is devoid of cytoplasmic inclusions. The B. mori hemocytes were morphologically distinct enough for identification under a phase-contrast microscope.
3. Results 3.1. Survival duration and mitotic division of prohemocytes in vitro Over 94% of the prohemocytes individually cultured in the basic medium survived the first 24 h of culture, but none of them underwent division. The survival rate and ratio of dividing cells were improved by adding larval hemolymph to the medium. When the medium contained 10% larval hemolymph, 8.1% of the cells underwent mitotic division during the first 24 h of culture (Fig. 2). At 50% hemolymph, this ratio increased to 10.7%. The average survival of dividing prohemocytes was 9.7–12.6 days, which was longer than that of non-dividing cells. By the end of culture, approximately 20% of prohemocytes that were incubated in medium containing hemolymph underwent mitotic division, compared with 1.8% in basic medium alone (Table 1). Among the prohemocytes cultured in medium containing
We examined the morphological changes undergone by cultured prohemocytes. Thirty two percent of nondividing prohemocytes cultured in basic medium differentiated to granulocytes (Table 2). The differentiation occurred 1–8 d of culture (3.5 d on average). The first phase of differentiation was an appearance of small granules in cytoplasm, giving rise to a round-shaped granulocyte containing many prominent granules, identical to the granulocyte just collected from the hemocoel [Fig. 3(a)]. The granulocytes extended filopodia 1–3 d after differentiation [Fig. 3(b)], and fully spread to adhere to the substratum 3–5 d after differentiation [Fig. 3(c)]. Thirty five percent of non-dividing prohemocytes differentiated to plasmatocytes (Table 2). The differentiation occurred following 1–7 d of culture (2.5 d on average). These cells first became spindle-shaped or oval cells without cytoplasmic inclusions, identical to the plasmatocytes just collected from the hemocoel [Fig. 3(e)]. The plasmatocytes adhered to the substratum 1–2 d after differentiation [Fig. 3(f)], and then became amoeboid with lamellopodia which are exclusive features of plasmatocytes 2–3 d after differentiation [Fig. 3(g)]. Eventually 67.9% of the prohemocytes differentiated and the remainder did not. The differentiated cells survived for over 2 days after differentiation. These results indicate that B. mori prohemocytes circulating in the blood can generate differentiated progeny such as plasmatocytes and granulocytes. Among the prohemocytes that mitotically divided in medium containing larval hemolymph, 59.2% of daughter cells became other types of hemocytes such as plasmatocytes, granulocytes and spherulocytes, whereas the remaining 40.8% divided into new prohemocytes (Table 3). Approximately 55% of the differentiation occurred 2–11 d after mitotic division, whereas 45% occurred within 24 h after division. Morphological changes of the differentiated hemocytes and the rate were essentially the same as those of the hemocytes differentiated from non-dividing prohemocytes. The composition of differentiated daughter cells indicated that 55.3% of cell divisions were symmetrical (single-type) and the remainder were asymmetrical (mixed-type). Among the asymmetrically divided prohemocytes, four cells gave rise to daughter cells composed of plasmatocytes and granulocytes, suggesting that these types of hemocytes form cells of the same lineage. These results showed that each prohemocyte can differentiate into different types of hemocytes, suggesting that the fate of B. mori prohemocytes is not predestined, even after dispersion from hematopoietic organs. Forty percent of prohemocytes were renewed by a symmetrical or asym-
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Fig. 2. Mitotic division of isolated prohemocyte in vitro. (a) Isolated prohemocyte, (b) mitotic phase of the prohemocyte, after 1 d of culture, and (c) two daughter prohemocytes, which separated within 1 h after mitotic phase. Scale bar=10 µm. Table 1 Division and survival of prohemocytes (PR) cultured in MGM-450 medium containing various concentrations of hemolymph ConcentrationTotal no. of % Divisiona Survival of hemocytesa of PR cultured (days) hemolymph (%)
0 10 50
92 99 74
1.8±1.8 19.6±0.8b 22.1±1.7b
Nondividing
Dividing
4.2±1.1 5.3±1.0 6.8±1.4
11.0 9.7±3.8 12.6±2.5c
from L5D2, L5D4 and L5D6 larvae supported 68.5, 60.2 and 28.3% differentiation, respectively (Fig. 4). Under each of these conditions, plasmatocytes and granulocytes were evident as differentiated types of hemocytes. When L5D2 or L5D4 larval hemolymph was added to the medium, the fate of differentiated prohemocytes was granulocyte-biased or non-biased, respectively. 4. Discussion
Values are means of three replicates, ±SD. Significantly higher than value for 0% hemolymph, P⬍0.05, ANOVA. c Significantly longer than period for non-dividing hemocytes, P⬍0.05, ANOVA. a
b
Optical and electron microscopic studies have provided valuable insight into the process of hemocyte differentiation. Devauchelle (1971) observed many intermediate stages among Melolontha melolontha prohemocytes, plasmatocytes, granulocytes and adipohemocytes, suggesting that each type is derived from the other. An ultrastructual and cytological study by Beaulaton and Monpeyssin (1976) suggested that pro-
Table 2 Frequency of cell types (%) differentiated from non-dividing prohemocytes cultured in medium containing various concentrations of hemolympha Concentration of hemolymph (%)
Total no. of PR cultured
PR (non-differentiated) PL (%) (%)
GR (%)
Total % of differentiated PR
0
92 79
50
57
32.5±0.4 (2.7±0.6) 26.1±4.8 (3.5±1.8) 19.6±0.7 (5.5±3.1)
67.9±0.5
10
32.1±0.3 — 40.8±3.1 — 49.8±1.7 —
a b
35.4±1.5 (2.2±0.2)b 33.2±0.1 (2.6±0.7) 26.8±2.3 (2.4±1.0)
59.2±3.1 46.4±1.9
Values are means of three replicates, ±SD. PR, prohemocyte; PL, plasmatocyte; GR, granulocyte. Values in parentheses are survival of differentiated cells (days).
metrical type of mitotic division. Conversely, plasmatocytes and granulocytes that differentiated from prohemocytes in vitro and which did not yet extend filopodia or protrusion, symmetrically divided to generate only the same types of hemocytes (Table 3). 3.3. Effect of hemolymph obtained from different stages of larvae on prohemocyte differentiation in vitro Larval hemolymph supported the differentiation of prohemocytes in vitro. However, hemolymph samples
hemocytes are stem cells that become plasmatocytes after several intermediate stages. Akai and Sato (1971) indicated that several types of differentiated B. mori hemocytes are released from hemopoietic organs through openings in the surrounding acellular membrane. Han et al. (1998) examined hemocytic differentiation in the hemopoietic organs of B. mori larvae, using transmission electron microscopy. Their study indicated that immature and mature hemocytes co-exist. They suggested that the lineage of each type of hemocyte involving prohemocytes in hemolymph can be formed from pluripotent stem cells located in compact islets of hemo-
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Fig. 3. Morphology of different types of hemocytes differentiated from isolated prohemocytes in vitro. (a) Non-adherent granulocyte, after 2 d of culture, (b) granulocyte, which protruded filopodia, 1 d after differentiation, (c) fully spread granulocyte, 2 d after differentiation, (d) spherulocyte, (e) non-adherent oval plasmatocyte, after 2 d of culture, (f) plasmatocyte, 1 d after differentiation, and (g) fully spread plasmatocyte, 2 d after differentiation. Scale bar=10 µm. Table 3 Composition of daughter cells from hemocytes cultured in medium containing 10 or 50% hemolymph Cell type at divisiona
Total no. of dividing hemocytes
Composition of daughter cells
(Single-type)
PR
38
(Mixed-type)
PR PR
PL PL
GR GR
SP SP
PR PL
PR GR
9
2
9
1
7
6
2.3c (1.2)d GRb
5
PLb
2
a b c d e
7.6 (3.6) 5e 5.8
7.0 (1.0)
3.3 (2.4)
3.2 (5.0)
PL GR 4 PL 6.8 (1.8)
GR 5.0 (6.8)
2 7.3
PR, prohemocyte; PL, plasmatocyte; GR, granulocyte; SP, spherulocyte. Hemocytes differentiated from prohemocytes in culture. Values below number of pairs of dividing hemocytes are survival of differentiated cells (days). Values in parenthesis are days from mitotic division to differentiation. Two of these subsequently transformed into spherulocytes before protrusion of filopodia.
poietic organs, and that the formed hemocyte may not undergo further transformation. These studies have provided significant information regarding hemopoiesis, but did not successively follow the fate of specific hemocytes. In the present study, we isolated and cultured individual B. mori prohemocytes from larval hemolymph. When cultured in MGM-450 medium without FBS, 1.8–22.1% of the prohemocytes underwent cell division. Furthermore, many prohemocytes directly obtained from hemolymph or produced by cell division of individually cultured prohemocytes differentiated into granulocytes, plasmatocytes and spherulocytes. Thus, the prohemocytes released from the hemopoietic organs and circulating in the blood are also pluripotent. Gupta and Suther-
land (1966) observed the in vitro transformation of plasmatocytes into various cell types such as granulocytes, spherulocytes and oenocytoids. Mitsuhashi (1966) reported that various cells intermediate between prohemocytes and plasmatocytes appeared in Chilo suppressalis hemocyte cultures, suggesting the transformation of prohemocytes into plasmatocytes. Sohi (1971) also suggested from an in vitro study that plasmatocytes are derived from prohemocytes. In the present study, several prohemocytes divided twice and some of the daughter cells gave rise to granulocytes after the second division (data not shown). Moreover, four prohemocytes produced daughter cells composed of plasmatocytes and granulocytes. These findings demonstrated that B. mori prohemocytes can self-renew and are pluripotent, sug-
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Fig. 4. Types of hemocytes differentiated from prohemocytes cultured in medium containing 10% hemolymph collected from various ages (L5D2, L5D4 and L5D6) of fifth instar B. mori larvae.
differentiation depended upon the stage of larvae from which it was obtained. These findings suggested that specific stimulants and/or materials are required to support hemocyte development in vitro. Many earlier studies of B. mori (e.g., Nittono, 1960) have noted that differential and total hemocyte counts fluctuate with the molting cycle, suggesting that hemopoiesis and the activity of hemocytes are under endocrine control. Hoffmann and Joly (1969) and Hoffmann (1970) found that the corpora allata influences both the differentiation and production of hemocytes in Locusta migratoria. Han et al. (1995) reported that 20-hydroxyecdysone (20OHE) and juvenile hormone, particularly 20-OHE, activates the discharge of hemocytes from hemopoietic organs in B. mori. Thus, the effects of insect hormones on prohemocyte differentiation should be examined.
Acknowledgements gesting that they have features characteristic of stem cells. However, oenocytoids were not differentiated from prohemocytes in the present study, although the former are a type of hemocyte in B. mori. These results suggested that the lineage of oenocytoids differs from that of other types of hemocytes. Thus, this study indicated that various types of hemocytes, with the exception of oenocytoids, can be generated by differentiation of circulating prohemocytes in addition to hemopoiesis in hemopoietic organs. Plasmatocytes and granulocytes predominate in B. mori hemolymph, and are associated with cellular defence against invading pathogens or parasitoid eggs, as well as the disposal of waste cells produced through larval development and metamorphosis. This study showed that these cells also undergo mitotic division in vitro and give rise to their own types of daughter cells. However, some of the granulocytes subsequently transformed into spherulocytes. Cockroach granulocytes differentiate into spherulocytes (Gupta and Sutherland, 1967). These facts indicate that granulocytes are not terminally differentiated. Granulocytes may be heterogeneous with respect to their fate, since the cells can be immunologically grouped into several sub-types (Brehelin and Zachary, 1986). Gardiner and Strand (1999) suggested using monoclonal antibodies against Pseudoplusia includens hemocytes to show that granulocytes are antigenetically related to spherulocytes. The present study showed that plasmatocytes and spherulocytes of B. mori may be terminally defferentiated cells, but granulocytes are not. Further studies are needed to determine whether B. mori granulocytes are heterogeneous with respect to their fate, or whether they are a transient form of spherulocytes. The survival and development of cultured prohemocytes were improved by including larval hemolymph in the medium. The effect of hemolymph on growth and
We are grateful to Dr H. Fugo for providing silkworm stocks.
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