Hemocytes of Penaeid and Palaemonid shrimps: Morphology, cytochemistry, and hemograms

JOURNAL

OF INVERTEBRATE

Hemocytes

PATHOLOGY

53, 64-77 (1989)

of Penaeid and Palaemonid Shrimps: Cytochemistry, and Hemograms

Morphology,

ANGBLIQUE TSING ERPAM, Laboratoire

de Pathologie Cornparke (CNRS), LJSTL PI. E. Bataillon,

34040 Montpellier,

France

JEAN-MICHEL ARCIER Laboratoire

de Physiologie des Invertebres, USTL, Montpellier,

France

AND

MICHEL BREHBLIN~ ERPAM, Laboratoire

de Pathologic Comparee (CNRS), USTL Pl. E. Bataillon, 34060 Montpellier,

France

Received December 8, 1987; accepted June 3, 1988 Hemocytes of Penaeus japonicus, Penaeus monodon, Macrobrachium rosenbergii, and Palaemon adspersus were separated into three cell types because of their ultrastructural features. These four crustacean species possessed hemocytes with small granules and hemocytes with large granules. In addition, hemocytes with a low level of differentiation were observed in the blood of P. japonicus and P. monodon. In P. monodon and M. rosenbergii the hemocytes with large granules were of two different subtypes due to the aspect of their inclusions. Some of the hemocytes with small granules, in P. adspersus, possessed vesicles which fused in large vacuoles and were not observed in the other species. In P. japonicus an acid phosphatase activity has been evidenced in granular hemocytes, especially in those with small granules. A phenoloxidase activity was confined to the cytosol of liemocytes with large granules. The three blood cell types of P. japonicus exhibited a glycocalyx stained with ruthenium red. Their plasma membrane possessed receptors for Con A, which showed different distributions because of the cell type. All these hemocyte types have been identified in light microscopy and the evolution of hemogram studied in an intermolt. B 1989 Academic

Press, Inc.

WORDS: Hemocytes; phenoloxidase; glycocalyx; hemogram; crustacean; Penaeus japonicus; Penaeus monodon; Macrobrachium rosenbergii. KEY

INTRODUCTION Rearing of economically important crustacean species (especially shrimps) is now conducted in a large scale in more and more numerous farms. The important concentrations of animals imposed by an intensive culture have triggered the development of epizootics (Bonami, 1976; Couch, 1978; Lightner et al., 1983a, b) which are often explosive and sometimes lead to the loss of the whole stock. The health condition monitoring of the populations under rearing is not yet very easy because techniques to make this control are missing. ’ To whom correspondence should be addressed.

and reprint requests

The study of hemocytes and hemograms is one possibility to perfect such techniques: it has been shown that infectious diseases reflect back on the blood of crustaceans (de Backer, 1961; Bang, 1971). Unfortunately, studies on modifications of the blood picture ( = hemograms) are very few. In fact, only one work has been published about the evolution of hemogram in the course of development (Bauchau and Plaquet, 1973, in the crab Eriocheir sinensis). Such investigations are missing for rearing shrimps which is why we studied hemocytes and hemograms and present our first results here. They were performed on Penaeus japonicus and Penaeus monodon (Penaeids) and Palaemon adspersus and 64

0022-2011/89 $1.50 Copyright 8 1939 by Academic Press. Inc. All rights of reproduction in any form reserved.

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HEMOCYTES

rosenbergii (Palaemonids) . Using light microscopy, several classifications of crustacean hemocytes have been settled, each author often utilizing various criteria of differentiation and an original terminology, and this has led to the multiplication of names making comparisons very difficult, as underlined by Rabin (1970). The works conducted under electron microscopy have shown that the crustacean hemocyte classification would be more unitary (Bauchau and Mengeot, 1978) than it appears in previous studies. But even with the help of ultrastructural studied, the classification of crustacean hemocytes is the subject of discussions (see the review by Bauchau, 1981, and the recent works of Martin and Graves, 1985; Benjamin and James, 1987). So, before following the hemogram modifications in the course of an intermolt, we studied the hemocyte ultrastructure and some parts of its cytochemistry and substantiated a classification of shrimp hemocytes in light of our results compared to previous studies. Macrobrachium

MATERIALS

AND METHODS

Animals

The shrimps used in these experiments were l-year-old adults and (or) 35 to 150 days old postlarvae. P. japonicus were provided by farms of the French Mediterranean coast, while samples of P. monodon and M. rosenbergii were reared in Tahiti (South Pacific). P. adspersus were caught in the Thau pond (Herault, France). In the laboratory, postlarvae were reared in 50liter seawater tanks (50 animals per tank) and fed daily with frozen Artemia nauplii. Adults were kept in 500-liter tanks (with a bottom of sand for P. japonicus) and fed with mussels twice a week. The stages of the molt cycle of postlarvae were determined according to Drach and Tchemigovtzeff (1967) adapted for penaeid shrimps (Cognie, 1970; Bourguet and Exbrayat, 1977). As a function of the develop-

h5

ment of uropod setae, four stages were distinguished: A, B (1 and 2), C, and D (1 to 4). We emphasize that making the distinction between D3 and D4 was sometimes impossible. So, in our results, we do not make a distinctive D3 stage and have regrouped some animals in D4 stage with those in D3. Animals which exhibited characteristic features of D4 stage were regrouped in a separate D4 stage. Numerations

Total hemocyte counts (THC) were performed in a Piette cell (depth = 0.02 mm) without diluting blood. Wet unfixed coverslipped whole amounts of hemolymph from postlarvae were examined under phase contrast microscopy for differential hemocyte counts (DHC). Statistical comparisons were made using the t test and F test for percentages (Snedecor and Cochran. 1957). Blood Collecting for Microscopy

and Preparation

For studies in light microscopy, hemolymph was removed from the cephalothorax with a Pasteur pipette. Hemocytes were either directly observed with phase contrast microscope or fixed in 2.5% glutaraldehyde in PBS (phosphate-buffered saline) immediately at blood removal. For transmission electron microscopy (TEM), after sectioning the dorsal vessel, hemolymph was collected directly in a 3% glutaraldehyde solution in cacodylate buffer. The fixed hemocytes were pelleted, washed in cacodylate buffer, postfixed in 1% osmium tetroxide in the same buffer, dehydrated, and embedded in Epon 812. Thin sections were contrasted according to Reynolds (1963) and examined with Hitachi HU 11B or JEOL 200CX electron microscopes at 75 or 80 kV. Cytochemical

Analysis

Acid phosphatase in hemocyte inclusions was searched for, using the method described by Essner (1973) with Na-Bglycerophosphate as substrate and cacodyl-

66

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ate and then Tris-maleate as buffers. In controls, the substrate was omitted. Phenoloxidase activity was studied in hemocytes, following the method described by Delachambre (1973) with tyrosine or DOPA (dihydroxyphenyl-alanine) as substrate. Ultrathin sections were not contrasted. In controls incubations were performed in the presence of phenylthiourea (PTU). Existence of a glycocalyx on the outer surface of hemocyte plasma membrane was pointed out, using ruthenium red as dye (Luft, 1971), which was added to the fixatives. Ultrathin sections were contrasted with uranyl-acetate alone. Some of the carbohydrate moieties of this outer layer have been determined according to their affinities for lectins. In a first step, hemocytes were fixed in a solution of 2.5% glutaraldehyde in phosphate buffer. They were then rinsed two times in the same buffer and three times in buffer plus glycine (0.15%). Hemocytes were incubated for 15 min in a solution of biotinylated lectin (0.2 mg/ml), rinsed four times in phosphate buffer, and incubated for 15 min in a ferritin-avidin solution (0.2 mg/ml). Finally, they were rinsed three times in phosphate buffer, posttixed in osmium tetroxide, dehydrated, and embedded as above. Lectins used were concanavalin A (Con A) and wheat germ A (WGA). For controls, hemocytes were incubated in lectin plus specific saccharide competitor (0.4 M) in place of lectin alone (D-mannose for Con A and N-acetyl-glucosamine for WGA). All these reactives were obtained commercially from Vector Laboratories. RESULTS

As already reported it was difficult to obtain a good definition of hemocytes in light microscopy. That is why we began this morphological study by the description and the definition of hemocyte types in electron microscopy. In a second step we searched for differences in some enzymes and cell coat composition between the cellular types previously defined on cytological fea-

AND

BREHkLIN

tures. These different cell types were finally identified in light microscopy, which allowed us to study the hemogram modilications in the course of an intermolt. Hemocyte

cytology

Because of their ultrastructural features, most of the hemocytes of P. juponicus can be divided into three main types (Fig. 1). Cells of the first type are elongated (8.6 x 3.5 pm). Most of the nuclear chromatin is dispersed (= euchromatin) (Fig. 1). In the cytoplasm, free ribosomes are numerous and the rough endoplasmic reticulum (rer) is moderately developed. Dictyosomes are few. These cells sometimes contain very rare little rounded cytoplasmic granules (less than 0.1 p,rn in diameter) which are membrane limited and uniformly opaque to electrons. As these hemocytes do not show pronounced signs of differentiation, we named them undifferentiated hemocytes (UH) (Fig. 2). The hemocytes belonging to the second type are irregular in shape (with an average of 9.5 x 5.7 km), with a lot of digitations (Fig. 3). They are characterized by the presence of cytoplasmic granules which are much more numerous than in the first hemocyte type described, and always of a spherical shape. These inclusions are surrounded by a membrane and their diameter varies from 0.09 to 0.3 pm. The smallest ones are always in the vicinity of a Golgi complex secreting small vesicles with an electron dense content. Free ribosomes are abundant and the rer is developed in narrow but elongated cisternae. The mitochodria are rounded and numerous, especially around the nucleus. The nucleus is smaller than that of LDH and often in horseshoe shape. Regions of very dense chromatin (= heterochromatin) are numerous and packed around the inside of the nuclear envelope. We call these cells small granule hemocytes (SGH). Hemocytes belonging to the third type are ovoid and about 10 x 7.0 pm (Fig. 4). Their oval nucleus occupies only a small

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HEMOCYTES

FIG. 1. Hemocytes of Penaeus japonicus. The blood has been directly collected in glutaraidehyde solution. Compare the aspect of chromatin in UH and in SGH and LGH. SGH show several pseudopods (arrowheads). 1, UH; 2, SGH; 3, LGH. TEM x6400.

FIG. 2. UH of Penaeusjaponicus. The nucleocytoplasmic ratio is high. Inclusions (arrow) are very rare in the cytoplasm. TEM x 12,000. FIG. 3. SGH of Penaeus japonicus. Small rounded dense granules are numerous. Dense chromatin is packed along the cistema in the horseshoe nucleus. *Pseudopod. TEM x9750. FIG. 4. LGH of Penaeus japonicus. The cytoplasm is filled with large dense granular inclusions. Cytoplasmic organelles (rer, mitochondria, dictyosomes) are scant. TEM x 12,000. FIG. 5. (a-e) Hemocytes of Penoeus japonicus in phase contrast microscopy. Hemolymph has not been collected in fixative. (a) UH: absence of inclusions and pseudopods. (b-c) SGH: in (b) very few inclusions are present whereas they are numerous in (c). (d-e) LGH: few inclusions in (d), numerous in (e). Note that the hemocyte types are easy to recognize because of the size and shape of granules rather than the number of them (light microscopy x 1125). FIG. 6. Second kind of LGH of Penaeus monodon. All the large granules (arrows) exhibit an internal structure. The cytoplasm has a very dark appearance. TEM x 15,000. Abbreviations used in Figures 2-11: d, dictyosome; LGH = large granule hemocyte; m, mitochondria; PO, phenoloxidase; rer, rough endoplasmic reticulum; SGH, small granule hemocytes; TEM, transmission electron microscopy; UH, undifferentiated hemocytes. Bar = 0.5 pm. 68

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part of the cellular volume and also possesses masses of dense heterochromatin close to the nuclear envelope. Hemocytes of this type are characterized by cytoplasmic granules often numerous and of different shapes: ovoid, spindle-shaped, but rarely spherical. They are always membrane bounded and generally possess a homogeneous electron-dense content; but, they sometimes exhibit an internal structure made of alternative electron dense and electron lucent bands. The diameter of these inclusions varies from 0.3 to 1.5 km. They seem to have issued from the fusion of dense vesicles synthesized by the Golgi apparatus. The cytoplasm of these cells contains few free ribosomes. Their rer is little developed and the mitochondria are scant. The hyaloplasm appears often finely granular. We named these hemocytes large granule hemocytes (LGH). These three hemocyte types are also present in P. monodon where a peculiar kind of LGH is observed (Fig. 6). In addition to the typical granules, these cells show large and elongated cytoplasmic inclusions, with a granular content. Their rer is well-developed and their cytoplasm is more electron dense than in other cells, giving them a very dark appearance. The blood of P. adspersus and M. rosenbergii lacks UH. These shrimps only possess granular hemocytes which are not different from those of P. japonicus. But in addition to the two typical granular hemocyte types, each palaemonid species exhibits a kind of granular hemocyte which shows peculiar features. In M. rosenbergii (Fig. 7), together with LGH, some blood cells show large and dense inclusions, very irregular in shape, never observed in the three other species. In some SGH of P. adspersus (Fig. 8), we observed numerous cytoplasmic vesicles, with granular contents weakly electron dense. These vesicles sometimes merge to form vacuoles as large as the nucleus. The origin of these inclusions is unknown. They seem not to have any relation with the rer or with the Golgi apparatus or

69

the dense granules. We emphasize that in the four crustacean species studied here, few sections of hemocytes containing small rounded granules (typical of SGH) together with large ones (typical of LGH) were observed. The following results are obtained only with P. japonicus hemocytes. Cytochemistry

The cytoplasm of SGH from P. japonicus exhibits vesicles with a positive reaction for acid phosphatase (Fig. 9a). When these vesicles are less than 0.5 p,rn in diameter, they are in general tilled with a dense precipitate. When they are larger (up to 1.2 pm) the positive reaction is confined to the inside of their membrane, with only some dots in other parts of the vesicles. The small vesicles originate from dictyosomes, in which the reaction for acid phosphatase is positive in the sole two or three last flattened cisternae of the maturing face (Fig. 9b). In LGH, vesicles with a positive reaction for acid phosphatase are much less numerous than in SGH, and almost absent in UH. The typical dense granules of LGH do not show any reaction for acid phosphatase under the conditions of our experiments. With the method used, a phenoloxidase activity could only be evidenced in the LGH, using DOPA as substrate (Fig. 10). The enzyme does not seem to be confined to a peculiar organelle but is diffused in the whole cytosol which shows a granular and electron-dense content. The typical granules of LGH do not show any positive reaction. This reaction is always negative for SGH and UH. It is also negative for LGH if tyrosine is used in place of DOPA as substrate. Using ruthenium red, a cell coat is easily evidenced (Fig. 11) and no marked differences are observed among the three hemocyte types previously described. Labelings with the two lectins used differ from one another. No labeling is observed after incubation in WGA. With Con A a gradation is observed from LGH to UH (Figs. 12 to 14). On the plasma membrane of LGH and

TSING,

ARCIER,

AND

BREHkLIN

FIG. 7. Second kind of LGH of Mucrobrachium rosenbergii. Dense granules are irregular in shape and sometimes of a large size (arrow). TEM x 12,000. FIG. 8. SGH of Pnlaemon adspersus. Some hemocytes of this type possess vesicles (asterisks) with a weakly electron dense content and which sometimes seem to fuse. TEM x 30,000. FIG. 9. Evidence for acid phosphatase in SGH of Penaeus japonicus. (a) Small granules are often uniformly dense whereas the largest ones are heterogeneous. Other granules do not show any positive reaction. Uncontrasted section. TEM x 19,500. (b) Dictyosome in SGH of P. japonicus. The sole last cistemae of the maturing face (arrowhead) and the secretory vesicle (arrow) are positive. Uncontrasted section. TEM x 19,500. FIG. 10. Study of phenoloxidase in hemocytes of Penaeus japonicus. The enzyme can be evidenced in cytosol of the sole LGH. Note that all the granular inclusions are negative for PO. Uncontrasted section. TEM X8250.

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HEMOCYTES

FIG. 11. Glycocalyx of Penaeus japonicus hem ocytes, stained with ruthenium red. Staining is the same for the three cell types. Note the long pro jections of the coat (arrowheads). Uranyl-acetate contrasted section. TEM x 15,000. FIGS. 12-14. Presence of receptors for Con A (on plasma membrane of Penaeus japonicus hemocytes. After fixation on the receptors, the biotinyl ated lectin is evidenced using avidin-ferritin. Note that ferritin shows a regular arrangement in UH (I jig. 12). in numerous bundles (arrowheads) in SGH (Fig. 13) and in rare bundles (arrowheads) in LGH (Fig. 14). Uranyl-acetate contrasted section. TEM x 30,000.

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AND

SGH, the labeling pattern exhibits a scattered distribution, but the amount of ferritin appears heavier on SGH than on LGH. On the plasma membrane of UH, the labeling pattern is more homogeneous and significant than in the two other hemocyte types. No labeling is observed when avidinferritin is used without any prior incubation in lectin or when D-mannose is added as inhibitor to the biotinylated Con A solution. Hemograms

In phase contrast microscopy, in a drop of hemolymph collected in fixative, the three cell types described above can be recognized by the size of their inclusions and their degree of refringency. The UH appear weakly refringent with some rare cytoplasmic granules. Their nucleus occupies the central part of the cell. The hemocyte with small granules are ovoid with some pseudopods and appear more refiingent than cells of the previous type. Cytoplasmic granules are evident but sometimes difficult to individualize because of their size; their nucleus is most often quite distinct. The size and sometimes the important number of cytoplasmic granules give a very refringent sight to hemocytes of the third type (LGH). Their nucleus is often invisible, hidden by the important refringenc y . In unfixed hemolymph, these three hemocyte types are also recognizable because of their inclusions (or absence of inclusions), but they appear to have many

TABLE EVOLUTION RELATIVE

Stages A-B 1 B2 C D2 D3-D4 D4

BREHl?LIN

shapes. UH (Fig. 5a) have a more or less rounded shape whereas SGH (Fig. 5b-c) and LGH (Fig. 5d-e) send out numerous pseudopods and lamellipodium. Moreover, in the course of hemocyte counts made on unfixed hemolymph, we observed some cells that we could not classify in one of the three types described above. In phase contrast microscopy, these cells are not very refringent. They are rounded in shape and their cytoplasm is only present as a thin rim all around an empty nucleus. In electron microscopy we attempted to recover this type of hemocytes by fixing hemolymph 10 min after its removal. In this way, we observed rounded cells with a nucleus devoid of dense chromatin and surrounded by a dilated cisternae, and which look like the hemocytes described by Bauchau and de Brouwer (1974) when the blood of Eriocheir sinensis coagulates. The transformations leading to these cells are described in detail elsewhere (A. Tsing and M. Brehelin, in preparation). In the present work we call these hemocytes hemocytes after natural lysis (HL). The studies on hemogram modifications, carried out on P. japonicus postlarvae, show that the number of cells per volume unit (THC, Table 1) varies as a function of the development stage. At the beginning of the cycle (stages A-Bl), this number is high. It decreases and reaches a minimum in B2. The difference between A-B1 and B2 is significant. Then the number of hemo-

1

OF THE TOTAL NUMBER OF HEMOCYTES PER CUBIC MILLIMETER OF BLOOD (THC), AND OF THE PERCENTAGE OF EACH HEMOCYTE TYPE IN AN INTERMOLT OF Penaeus japonicus POSTLARVAE (MEANS OF 10 ANIMALS AND STANDARD DEVIATIONS)

THC 11,500 4,900 7,600 8,400 14,600 5,400

+ 2 t * k 2

UH 5,500 2,000 3,100 3,000 7,100 2,500

8.9 10.3 10.9 10.6 9.1 10.8

f + + f 2 5

SGH 3.6 4.8 9.7 3.5 7.7 5.0

20.3 22.4 18.5 20.2 23.1 20.1

f k f k k *

8.4 10.8 12.3 6.5 11.0 15.4

LGH 15.4 14.6 12.6 19.6 15.9 13.8

t 2 * 2 * k

HL 12.4 12.7 5.9 8.9 10.9 11.9

55.4 52.7 58.0 49.6 51.9 55.3

k ” + + f 2

9.8 20.3 8.7 13.6 12.1 13.1

Note. The large amplitude of standard deviation in D3-D4 is due to the difficulty of determining the exact stage of development in some animals. HL = cells which lysis in vitro.

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cytes increases in stage C and remains stable in D2. It increases again and reaches a maximum in D3-D4. Augmentation of the THC between B2 and D3-D4 is significant. To make the differential hemocyte counts, we have taken into account four cell categories: undifferentiated hemocytes, small granule hemocytes, large granule hemocytes, and hemocytes after natural lysis. As the percentage of this last cell category increases with time, the differential hemocyte numerations are performed in the 10 min following the blood removal. The relative percentage of hemocytes is almost stable for the whole intermolt and for each cell type (Table 1). However, we have to underline the modifications observed between stages C and D for the hemocytes with large granules. In the course of this period, the percentage of LGH increases from 12.6 to 19.6 whereas the rate of HL decreases from 58 to 49.6%. Because of the weak variations noted in the DHC in the course of an intermolt cycle, evolution of the number of hemocytes per cubic millimeter is, for each cell type, nearly the same as that observed for total hemocyte counts (Fig. 15). DISCUSSION

The hemocytes of P. japonicus and P. monodon can be divided into three main categories: undifferentiated hemocytes (UH), hemocytes with small granules (SGH), and hemocytes with large granules (LGH). In P. adspersus and M. rosenbergii, the UH are lacking. This distinction in well-defined hemocyte types, evidenced under electron microscopy, can be found again in light microscopy where the differences in granule size are also visible. Whereas in P. japonicus all the LGH exhibit the same ultrastructural features, in P. monodon and M. rosenbergii hemocytes of this type seem to exist in two different forms. Because of the aspect of their large granules and nucleus and to the development of their rer, in a first approach we make the hypothesis that they are two physiological forms of a same cell type.

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12-s-

7.5 _ 5 2-5 ! FIG. 1.5. Evolution of the number of cells of each type per cubic millimeter of blood (ordinate) in the course of an intermolt (abscissa) in Penaeus japonicus postlarvae. Means of 10 animals and standard deviations. A-B1 to D4: stages of development. 0, hemocytes after natural lysis; W, small granule hemocytes; *, large granule hemocytes; 0. undifferentiated hemocytes.

The distinction into categories LGH and SGH is in good agreement with the work of Martin and Graves (1985) in the shrimps Sicyonia ingestis and Penaeus californiensis. The terminology chosen by these authors to name two of their cell types (LGH and SGH) has been assumed in our present work. In most of the other studies of crustacean hemocytes, conducted in electron microscopy, these blood cells were separated into three categories named hyaline, semigranular, and granular hemocytes (see review by Bauchau, 1981). This terminology refers to the number of granules for each cell type (Bauchau and Mengeot, 1978). In our study, it is more evident and easier to separate the hemocyte types using

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the size and shape of the granules rather than the amount of these inclusions in each cell. Moreover, the loads in granules refer to different levels of the same physiological activity of a cell, whereas the differences in shape and size of the secretions refer to different kinds of activity, which could more easily be connected with differences in cell types. Concerning the “hyaline hemocyte” of the literature (Bauchau, 1981) or the “agranular cell” of Martin and Graves (1985), we want to emphasize that this name is correct only in light microscopy where the cells of this type appear little contrasted and seem devoid of granular inclusions. In electron microscopy, hemocytes without any granules are very scant in the blood of P. japonicus and P. monodon, and even in the rare cases where they were seen (as one only observed a few sections of the same cell) it is not possible to affirm that this cell does not possess any granular inclusion. This is why we prefer not to use the names hyaline hemocyte or agranular cell. Other features of this hemocyte cytology, such as an important nucleocytoplasmic ratio, the large amounts of euchromatin, the little development of Golgi apparatus, as well as the high number of free ribosomes, make cells of this kind look similar to hemocytes at an early step of differentiation and allow us to name them undifferentiated hemocytes. In P. adspersus, they have never been observed in the blood, suggesting that differentiation take place elsewhere. Our conclusions on the absence of true agranular hemocytes is in agreement with those of Lockhead and Lockhead (1941), Hearing and Vernick (1967), Stang-Voss (1971), and Hoarau (1976). The UH are not to be confused with HL. These last cells are not present in the circulation. They only appear at the level of an injury or at blood removal (A. Tsing and M. Brehelin, in preparation). Most of our results on cytochemistry of P. japonicus blood cells support the separation of hemocytes into three distinct types as well as our terminology of undifferentiated hemocytes. We have been able

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to observe a positive reaction for phenoloxidase (PO) in the sole LGH. Although this seems to be the first attempt to prove the presence of this enzyme on an ultrastructural level in crustaceans, our result is in good agreement with those of authors working in light microscopy. Using several substrates and incubation methods different than ours, Decleir and co-workers (1960) have shown that PO is confined to L‘ . . . large oval cells with many large granules . . .” in Carcinus maenas. For these authors as for us, the reaction was positive with DOPA and negative with tyrosine. After separation of the hemocyte populations of C. maenas by density gradient centrifugation, Sdderhall and Smith (1983) also showed a PO activity in the sole “granular hemocytes,” most of them corresponding to our LGH. All these concordant results permit the aflirmation that PO activity is one of the functions sustained by LGH. Our work shows that this PO activity is not confined to cytoplasmic organelles or vesicles (and especially to the typical granules). Acid phosphatase is present in dictyosomes and vesicles of SGH and LGH, both exhibitinig lysosomial bodies, but the fact that this enzyme has been mainly observed in SGH could be related to the endocytotic capabilities of these cells (Brehelin and Artier, 1985). This is in opposition to the work of Benjamin and James (1987) in Ligia oceanica, but in agreement with the conclusive results of Smith and Ratcliffe (1978). In C. maenas, these authors demonstrated that phagocytosis is mainly achieved by “. . . phagocytic cells which had a variable number of small (less than 1.0 pm) intracellular granules. . . .” We think that the hemocytes described as phagocytic cells by Smith and Ratcliffe correspond to our SGH. Whereas the difference between hemocyte types was less obvious than for PO, the study of acid phosphatase also confirms the morphological separation we have made between SGH and LGH. Using ruthenium red, evidence for a cell coat on blood cells has been obtained in humans (Behnke, 1968), as well as in fresh-

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water molluscs (Sminia et al., 1981) and in insect (Beaulaton, 1985). We show here that hemocytes of marine animals also possess this glycocalyx. Unfortunately, there was no difference in ruthenium red affinity among the three hemocyte types previously described. In the same way any of these cell plasma membrane displayed WGA binding and all three bound Con A, although with different patterns. This suggests that all P. japonicus blood cells possess in their cell coat glucose or (and) mannose or (and) fructofuranosides residues acting as Con A receptors. In the glycocalyx, these receptors seem to have arrangements depending on the hemocyte type. These sites are the most numerous in UH. The hemocyte ultrastructure we describe can be observed in most of the decapod crustaceans studied to date. The main differences are with terminology (Bauchau and de Brouwer, 1972; Johnston et al., 1973; Hoarau. 1976; Bodammer, 1978; Martin and Graves, 1985; Benjamin and James, 1987) but also with possible affiliations between hemocyte types. The differentiation of crustacean hemocytes is the subject of discussions which remain open. Different authors have proposed an evolution from their “hyaline cells” toward their “granular hemocytes” (see, for instance, Bodammer, 1978; Bauchau and Mengeot, 1978; Benjamin and James, 1987) or in a reverse order starting from “granular hemocytes” toward “hyaline hemocytes” (Vranckx and Durliat, 1977). On the other hand, Sternshein and Burton (1980) failed to find evidence suggesting that, in crayfish, the observed hemocyte types are functional or developmental stages of one cell type. From our micrographs it is evident that some rare cells possess small granules together with large oval ones, suggesting a possible pathway from one hemocyte type to another. But to set up such a conclusion, special studies are needed. The results of hemocyte numerations that we obtained in the course of an intermolt do not allow us to answer. Works on morphology alone do not permit discussion about hemocyte affilia-

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tion and this is not the intention of the present work. In vitro studies, labeling of hemocytes with radioactive isotopes, and depression or stimulation of hemopoiesis are needed; but whatever the relations between the different hemocyte types, it is necessary to define precise cell categories in order to compare other works on functions or affiliation. In P. japonicus, the total hemocyte counts (THC) show two minima, one in B2 and the other in late D4. This second minimum is preceded by a high increase in hemocyte number in D3 and early D4. As the relative percentage of each category of cells does not show appreciable changes on these times, it is not possible to say if these maxima and minima are related to hemocyte production or disappearance, or to changes in the blood volume. There are very few studies on hemocyte numerations. Most of them are on the methods of sampling (Yeager and Tauber, 1935) or on the effects of diet or rearing conditions (Stewart et al., 1967), sex (Cornick and Stewart, 1978; Mix and Sparks, 1980), or pathogens (Sawyer et al., 1970; Mac Key and Jenkin, 1970; Smith and Ratcliffe, 1980; Dappen and Nickel, 1981) on the rate of circulating cells. To our knowledge, only one study (Bauchau and Plaquet, 1973) has taken into account variations of hemocyte counts m an intermolt. The main difference observed in the THC between the work by Bauchau and Plaquet in E. sinensis and ours is related to a peak in C stage, which we do not observe in P. &on&s. The other results are in good agreement. It is now possible to affirm that, in decapod crustaceans, exuviation is preceded by a fall of 50% of the total circulating hemocytes. In E. sinensis the THC varies from 9000 cells/mm3 (minimum) to 23,000 cells (maximum) and only from 9000 to 15,000 cells in P. juponicus. We think that the variations evidenced, which depend only on the stage in an intermolt, are important enough to be taken into account when studies on the modifications of hemocyte rate are needed, for instance, to study the role of these cells in the general

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physiology actions.

TSING,

ARCIER,

of crustaceans or in defense re-

ACKNOWLEDGMENTS We are indebted to Dr. M. Weppe for his help in the studies performed with P. monodon and M. rosenbergii. Work was supported by Grants 84LO913 from Minis&e de I’Industrie et de la Recherche and 521003 from IFREMER, France.

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