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First cytochemical study of Introduction Correspondence: Valerio Matozzo, Department of haemocytes from the Biology, University of Padova, via Ugo Bassi, aestuarii (Crustacea, Like other , Crustacea have an 58/B, 35131 Padova, Italy. ) open vascular system in which numerous Tel.: +39.049.8276201 - Fax: +39.049.8276199. haemocytes freely circulate in haemolymph. E-mail: [email protected] haemocytes are involved in impor- V. Matozzo, M.G. Marin Key words: Carcinus aestuarii, crab, haemocytes, tant functions, such as wound repair and Department of Biology, University of light microscopy, cytochemical assays, morpho- defence mechanisms against parasites, virus- logical characterisation. Padova, Italy es and bacteria.1 In many crustacean species, haemocytes originate from a specialised Received for publication: 1 December 2009. haematopoietic tissue generally situated on Accepted for publication: 14 January 2010. the dorsal and dorsolateral sides of the stom- Abstract ach and surrounded by connective tissue. This work is licensed under a Creative Commons Attribution 3.0 License (by-nc 3.0). Cells, perhaps haematopoietic cells, are For the first time, a morphological study of packed in small lobules, and some of these ©Copyright V. Matozzo and M.G. Marin 2010 haemocytes from the crab Carcinus aestuarii cells are also found in the interlobular spaces.2 Licensee PAGEPress, Italy was carried out by means of light microscopy Although the haematopoietic tissue has been European Journal of Histochemistry 2010; 54:e9 and differing cytochemical assays. Analysis of identified in some crustacean species, such as doi:10.4081/ejh.2010.e9 haemocyte size frequency distribution (per- , Homarus americanus, Pacifastacus leniusculus, and Penaeus mon- formed by means of a Coulter Counter) locytes), whereas in the blue crab, Callinectes odon,3-6 the relationship between haematopoi- revealed the presence of two distinct haemo- sapidus, three types of haemocytes – hyaline etic tissue cells and circulating haemocytes is cyte fractions in C. aestuarii haemolymph, cells, small and large granulocytes – were iden- still unclear. depending on cell size. The first fraction was of tified by Clare and Lumb.18 Surprisingly, eleven Classification of crustacean haemocytes has about 3-5 µm in diameter and 30-50 fL in vol- haemocyte types were recently described in the been (and still is) one of the most debated ume, the second was of about 6-12 µm in diam- American lobster, H. americanus, using mor- point, mostly due to the lack of uniform classifi- eter and over 200 fL in volume. Mean cell diam- phological criteria.19 cation criteria that enable to distinguish cell eter and volume were 8.20±1.7 µm and To our knowledge, no study concerning mor- types.1,2 Classification schemes are different for 272.30±143.5 fL, respectively. Haemocytes phological characterisation of circulating each species and are based on either haemo- observed under light microscope were distin- haemocytes from the crab Carcinus aestuarii cyte cytochemical properties, morphological guished in three cell types: granulocytes (28%; has previously been performed. As a conse- aspect, biological functions or observation tech- 11.94±1.43 µm in diameter) with evident cyto- quence, the aim of the present study was to niques. Therefore, resulting nomenclature is plasmic granules, semigranulocytes (27%; examine crab haemocytes under light micro- often controversial. Hose et al.7 and Gargioni 12.38±1.76 µm in diameter) with less granules scope and classify them by means of various and Barracco8 proposed to combine morpholog- than granulocytes, and hyalinocytes (44%; cytochemical assays, prior to perform both ical, cytochemical and functional features of the 7.88±1.6 µm in diameter) without granules. In ultrastructural and functional studies of crustacean haemocytes for a more convenient addition, a peculiar cell type was occasionally haemocytes. classification. In most of the Crustacea species, found (about 1%): it was 25-30 µm in diameter haemocyte classification is generally based on and had a great vacuole and a peripheral cyto- the presence/absence of cytoplasmic granules. plasm with granules. Granulocyte and semi- Following this criterion, three types of circulat- granulocyte granules stained in vivo with Materials and Methods ing haemocytes are usually recognised in Neutral Red, indicating that they were lyso- Crustacea: hyalinocytes (the smallest cells somes. Giemsa’s dye confirmed that granulo- without evident granules), semigranulocytes cytes and semigranulocytes were larger than (containing small granules) and granulocytes Intermoult adult males of C. aestuarii (4 cm hyalinocytes. Pappenheim’s panoptical stain- (with abundant cytoplasmic granules) (see mean carapace length) were collected by hand- ing and Ehrlich’s triacid mixture allowed to Bauchau1 for a review). However, other synony- made traps in the lagoon of Venice, kept in the distinguish granule-containing cells (includ- mous terms were proposed in the past to indi- laboratory in large aquaria provided with a ing semigranulocytes) in acidophils (64%), cate these cell types. For example, granulocytes sandy bottom, in seawater at salinity of basophils (35%) and neutrophils (1%). were also defined as granular eosinophils9 or 35±1‰, at a temperature of 17±0.5°C, and fed Hyalinocytes showed always a basophilic cyto- granular amoebocytes,10 semigranulocytes were with mussels (Mytilus galloprovincialis). The plasm. Haemocytes were positive to the PAS indicated as monocytes11 or intermediate cells12 were acclimatised in the laboratory for 5 reaction for carbohydrates, even if cytoplasm and hyalinocytes were also termed phagocytes13 days before analyses. carbohydrate distribution varied among cell or pro-haemocytes.14 Johnston et al.15 classified types. Lastly, lipids were found on cell mem- Carcinus maenas haemocytes in two types (α- brane and in cytoplasm of all haemocyte types and β-cells), whereas William and Lutz16 divid- Haemolymph sampling in the form of black spots produced after Sudan ed the haemocytes from the same crab species Crabs were anaesthetised on ice for 10 min, Black B staining. The morphological character- in two types, on the basis of the and the haemolymph (at least 250 µL/crab) isation of C. aestuarii haemocytes by light presence/absence of glycogen-containing gran- was collected from the unsclerotised mem- microscopy was necessary before performing ules. In the Indian spiny lobster, Panulirus brane of the walking legs using a 1-mL plastic both ultrastructural and functional studies of homarus, Manjula et al.17 observed four types of syringe, placed in Eppendorf tubes and diluted circulating cells. haemocytes (pro-hyalocytes, hyalocytes, eosi- 1:2 in an anticoagulant solution of citrate nophilic granulocytes and chromophilic granu- buffer/EDTA (NaCl 0.45 M, glucose 0.1 M, sodi-

[page 44] [European Journal of Histochemistry 2010; 54:e9] Original paper um citrate 30 mM, citric acid 26 mM, EDTA 10 FSW. Haemocytes were then observed in vivo, mM, pH 4.6) stored at 4°C.20 and lysosomes appeared red. Results ii) Giemsa’s dye. Haemocytes were stained for Haemocyte size frequency 10 min in a 10% Giemsa (Fluka) aqueous solu- Analysis of haemocyte size frequency distri- distribution tion, washed in distilled water and then bution revealed the presence of two main mounted. Nuclei appeared blue and cytoplasm haemocyte fractions in the haemolymph of C. Haemocyte size frequency distribution was pale blue or violet. aestuarii, depending on cell size (Figure 1A, determined immediately after haemolymph iii) Pappenheim’s panoptical staining. 1B). The first fraction was of about 3-5 µm in sampling by a Model Z2 Coulter Counter elec- Haemocytes were stained for 3 min in May- diameter and 30-50 fL in volume, the second tronic particle counter/size analyser (Coulter Grünwald’s dye (Fluka). After washing in dis- was of about 6-12 µm in diameter and over 200 21 Corporation, FL, USA). Briefly, 125 µL of tilled water, they were stained for 5 min in 5% fL in volume. Mean cell diameter was 8.20±1.7 haemolymph from 10 individual crabs were Giemsa, washed in distilled water and mount- µm, whereas mean cell volume was added to 19.875 mL of 0.45 µm filtered seawa- ed. Basophil granules appeared blue and aci- 272.30±143.5 fL (n=10). ter (FSW). Anticoagulant or fixative solutions dophils dark pink. Three main haemocyte types were identi- were omitted. Mean cell diameter and volume iv) Ehrlich’s triacid mixture. This mixture is fied in the haemolymph of C. aestuarii by light were measured and expressed as µm and fem- composed of 12 parts of saturated Orange G microscopy. In in vivo observation, cells were tolitres (fL), respectively. aqueous solution, 8 parts of saturated acid classified on the basis of the presence/absence fuchsin aqueous solution, 10 parts of saturated of cytoplasmic granules as granulocytes Short-term haemocyte cultures and methyl green aqueous solution, 30 parts of dis- (11.94±1.43 µm in diameter; n=100), with a in vivo observation of haemocyte tilled water, 18 parts of absolute ethanol and 5 great number of refractile granules (Figure parts of glycerine. Haemocytes were stained in Pools of haemolymph (from 2 crabs each) 2A), semigranulocytes (12.38±1.76 µm; this mixture for 15 min, washed in distilled n=100), containing a variable number of were used to prepare short-term haemocyte water and mounted. Basophilic granules cultures. Pooled haemolymph was then cen- refractile granules (Figure 2B) and hyalino- appeared pale green, neutrophilic violet and cytes (7.88±1.6 µm; n=100), without evident trifuged at 780¥ g for 10 min, the supernatant acidophilic appeared coppery red. cytoplasmic granules (Figure 2C). In addition, was discarded, and haemocytes were resus- v) Carbohydrate detection (Periodic Acid- pended in an equal volume of FSW. Short-term a peculiar cell type (25-30 µm in diameter), Schiff, PAS). A commercial Kit (Periodic Acid defined as lipo-protein cell, was occasionally haemocyte cultures were prepared according Schiff -PAS- Staining System, Sigma) was 22 found (about 1%) in haemolymph. It had a to Ballarin et al. : 60 μL of haemocyte suspen- used and manufacturer instructions were fol- sions were placed in the middle of culture great vacuole and a peripheral cytoplasm with lowed. Positive sites appeared pink/red. evident granules (Figure 2D). chambers made by glueing Teflon rings (15 vi) Lipid detection (Sudan Black B staining). mm internal diameter, 1 mm thickness) on After Giemsa staining, granulocytes (28%) Haemocytes, fixed as above, were washed in showed an oval, eccentric nucleus and abundant glass slides; a small amount of vaseline was PBS, treated for 3 min with 50% ethanol and put on the rings surface to allow adhesion of cytoplasmic granules (about 0.2-0.5 µm in diam- immersed for 15 min in a saturated solution of eter) (Figure 3A). Conversely, hyalino-cytes the coverslips. The culture chambers were Sudan Black B in 70% ethanol. Slides were (44%) had a large, central, round nucleus and then kept upside down for 30 min at room tem- then immersed in 50% ethanol, rinsed with did not contain granules appreciable under the perature to allow adhesion of the haemocytes distilled water and mounted. Black spot light microscope (Figure 3B, 3C). Semigranu- to the coverslips. For in vivo observation, revealed the presence of lipids. haemocytes were not fixed. The diameter of locytes (27%) were an intermediate cell type between hyalinocytes and granulocytes: they each haemocyte type was measured in 100 Image analysis cells by means of an image analysis software showed an eccentric, spherical nucleus and had (see below for details). Cell diameter was Light microscopic images were acquired by less granules than granulocytes (Figure 3A, 3C, measured in unfixed haemocytes to avoid a system consisting of a colour video camera 3D). Both granulocytes and hyalinocytes shrinkage due to fixation. (JVC 3-CCD), a Leica DM-LB microscope appeared as spherical cells (round haemocytes) (1000x magnification) and a PC provided with or amoebocytes (spreading haemocytes), being ImageNT and Microimage software developed able to emit pseudopodia. Granulocytes and Cytochemical assays by CASTI Imaging (Italy). Each assay was semigranulocytes (but not hyalinocytes and lipo- Except for the in vivo Neutral Red assay, repeated in triplicate. protein cells) stained in vivo with Neutral Red haemocyte monolayers were fixed for 30 min at 4°C in a solution of 1% glutaraldehyde (Fluka) in FSW containing 1% sucrose. They were then washed in phosphate buffered saline, pH 7.2, (PBS: 1.37 M NaCl, 0.03 M KCl,

0.015 M KH2PO4, 0.065 M Na2HPO4) and stained according to various cytochemical methods in order to identify haemocyte types. Slides were mounted in Acquovitrex (Carlo Erba, Milan, Italy) and observed under a light microscope. The following cytochemical stain- ing methods were used: i) Neutral Red dye. After haemocyte adhesion to the coverslips, FSW was removed from the culture chambers and substituted with 60 L Figure 1. An example of haemocyte size frequency distribution in C. aestuarii. Haemocyte μ diameter (A), expressed in µm, and haemocyte volume (B), expressed in femtolitres (fL). of a 8 mg/L Neutral Red (Merck) solution in

[European Journal of Histochemistry 2010; 54:e9] [page 45] Original paper dye, indicating that stained granules were lyso- somes (Figure 3E, 3F). Granule-containing cells (including semi- granulocytes) were further divided into three different subpopulations: acidophils (64%), basophils (35%) and neutrophils (1%) (Figure 4A-4F). Basophils had a blue (Pappenheim's staining) or pale green (Ehrlich's mixture) cytoplasm, acidophils showed a reddish-pink (Pappenheim) or coppery red (Ehrlich) cyto- plasm, while neutrophils had a violet cyto- plasm (Ehrlich). Hyalinocytes showed always a basophil cytoplasm. In C. aestuarii, all haemocyte types were positive to the PAS reaction, revealing the presence of carbohydrates (Figure 5A-5C). However, carbohydrate distribution differed among cell types. In granulocytes (Figure 5A), carbohydrates diffused throughout the cyto- plasm and inside granules, in hyalinocytes (Figure 5B) carbohydrates diffused throughout the cytoplasm, whereas in semigranulocytes (Figure 5C) PAS positivity was confined to the cytoplasm periphery and not in granules. Figure 2. Unfixed haemocytes of C. aestuarii, observed in vivo (A-D). g: granulocyte; sg: All cell types were positive to the Sudan semigranulocyte; h: hyalinocytes; lp: lipoprotein cell. Bar length: 5 µm. Black B staining for lipids. Lipids were mainly detected on cell membranes, even if black spots were also observed in the cytoplasm of granulocytes (Figure 5D-5F).

Discussion

In spite of the efforts of some authors (see Bauchau,1 for example) to establish a conven- ient terminology for decapod haemocytes, their classification is still controversial. This is mainly due to differing criteria used to classify crustacean haemocytes. As a consequence, confusion in nomenclature arises and compar- ison of blood cells from differing crustacean species is often difficult. In the present study, we examined under light microscope the circulating haemocytes from the crab C. aestuarii and proposed their classification on the basis of both morphologi- cal (i.e., presence/absence of cytoplasmic granules) and cytochemical features of cells. Analysis of haemocyte size frequency distribu- tion, never performed in a crab species, revealed the presence of two distinct haemo- cyte fractions in C. aestuarii haemolymph. They ranged from 3-5 µm (the first) and from 6-12 µm (the second). The Coulter Counter analysis provided a general view of haemocyte populations and suggested that C. aestuarii haemocytes had a great variability in cell size. After this preliminary analysis, C. aestuarii haemocytes were examined under light micro- scope in order to distinguish cell types. Figure 3. C. aestuarii haemocytes stained with Giemsa's dye (A-D) and vitally stained Interestingly, cell size measured in unfixed with Neutral Red, showing lysosomes (arrows) (E, F). g: granulocytes; sg: semigranulo- haemocytes using the ImageNT and cytes; h: hyalinocytes; lp: lipoprotein cells. Bar length: 5 µm.

[page 46] [European Journal of Histochemistry 2010; 54:e9] Original paper

Microimage software (granulocytes: 11.94±1.43 µm in diameter; hyalinocytes: 7.88±1.6 µm; semigranulocytes: 12.38±1.76 µm) was almost similar to that recorded by the Coulter Counter. Indeed, two main fractions of haemocytes were detected under light micro- scope: the former of about 6-7 µm, the latter of about 11-12 µm. Following the morphological criterion proposed by Bauchau1 and Hose et al.,7 that is the presence/absence of refractile granules, three basic cell types were recog- nised in the present study: granulocytes, sem- igranulocytes and hyalinocytes. A similar clas- sification was also adopted for haemocytes from other Crustacea species, such as Panulirus cygnus, Potamon fluviatilis and Liocarcinus depurator.23-25 Gargioni and Barracco8 classified haemocytes from both the penaeid, Penaeus paulensis, and the two palae- monids, Macrobrachium acanthurus and M. rosenbergii, as hyaline haemocytes, small granule haemocytes (semigranulocytes, in the present study) and large granule haemocytes (granulocytes, in the present study). Likewise, after Wright-Giemsa staining and observation under the transmission electron microscope, circulating haemocytes from the Chinese prawn Fenneropenaeus chinensis were divided into hyaline haemocytes, small granular haemocytes and large granular haemocytes.26 Conversely, in Cancer pagurus, Vogan and Rowley27 identified four haemocyte types – hyaline cells, eosinophil granulocytes, basophil granulocytes, eosinophil/basophil granulocytes – on the basis of nucleus morphology and dye- ing characteristics of cytoplasm. Figure 4. C. aestuarii haemocytes stained with Pappenheim’s (A-D) and Ehrlich’s triacid 7 mixtures (E, F); h: hyalinocytes; Ag: acidophil granulocytes; Bg: basophil granulocytes; Although Hose et al. used the location of the Ng: neutrophil granulocytes. Bar length: 5 µm. nucleus (central or eccentric) as a factor to distinguish small granule haemocytes (semi- granulocytes in the present study) from large granule haemocytes (granulocytes in the pres- ent study), we suggested that nucleus position is not an useful criterion to distinguish gran- ule-containing cells in C. aestuarii. Indeed, in this crab species, granulocytes and semigranu- locytes showed generally an eccentric nucleus. Hyalinocytes were morphologically different from granulocytes and semigranulocytes when examined under the light microscope. They were smaller than granule-containing cells, had a higher nucleus:cytoplasm ratio, a basophilic cytoplasm and did not contain evi- dent granules. In the present study, both gran- ulocytes and hyalinocytes of C. aestuarii showed round or amoeboid shape, haemocytes Figure 5. C. aestuarii haemocytes stained with PAS (A-C) and Sudan Black B (D-F). Red being able to emit pseudopodia. Round and arrows indicate black deposits on cell membrane and in cytoplasm; g: granulocytes; sg: spreading haemocytes were also observed in semigranulocytes; h: hyalinocytes. Bar length: 5 µm. Penaeus japonicus, even if in granulocytes only.28 In C. aestuarii, granulocyte cytoplasm was packed with large, round, refractile gran- Ehrlich’s triacid mixture, granule-containing Interestingly, a peculiar cell type was occa- ules, while semigranulocytes contained less cells (including semigranulocytes) of C. aestu- sionally found in haemolymph from C. aestu- granules than granulocytes. By means of both arii were further divided into acidophilis arii. It showed a great vacuole, a peripheral Pappenheim’s panoptical staining and (more than 65%), basophils and neutrophils. nucleus and a cytoplasm with evident gran-

[European Journal of Histochemistry 2010; 54:e9] [page 47] Original paper ules. We assume that it is not a true haemo- the cell membrane of both hyalinocytes and 1995;4:59-70. cyte, being rarely detectable in haemolymph. A granule-containing cells. However, black 6. Van de Braak CBT, Botterblom MHA, Liu W, similar cell type has previously been described deposits were also found in the cytoplasm of et al. The role of the haematopoietic tissue by Sewell29 in haemolymph from the closely granulocytes. In H. americanus, P. interruptus, in haemocyte production and maturation related species, C. maenas. In that study, the and L. grandis, only hyalinocytes were positive of the black tiger shrimp (Penaeus mon- author named it “lipo-protein cell” and sug- to Sudan Black B.7 In that study, granules of the odon). Fish Shellfish Immunol 2002; gested that its presence in haemolymph was hyaline cells from all the three decapod species 12:253-72. related to the moult stages of crabs (mainly at did not accumulate the stain, although mem- 7. Hose JE, Martin GG, Gerard AS. A decapod the C4 stage end), these cells being responsi- branes around the larger granules in some hemocyte classification scheme integrat- ble for the secretion of the non-chitinous epi- hyalinocytes of H. americanus displayed ing morphology, cytochemistry, and func- cuticle prior to moulting.29 In order to confirm intense staining.7 Conversely, granulocyte tion. Biol Bull 1990;178:33-45. the hypothesis by Sewell,29 future studies will cytoplasm remained unstained by Sudan Black 8. Gargioni R, Barracco MA. Hemocytes of be performed in C. aestuarii to establish a rela- B and the nucleus was always visible.7 the Palaemonids Macrobrachium rosen- tionship between crab moult stages and the Although the present study is not aimed to bergii and M. acanthurus, and of the presence of this peculiar cell type in haemo- discuss the hypothetical differentiation path- Penaeid Penaeus paulensis. J Morphol lymph. ways of C. aestuarii haemocytes (see Bauchau1 1998;236:209-21. As for the abundance of each haemocyte for hypotheses on crustacean haemocyte dif- 9. Cornick JW, Stewart JE. Lobster (Homarus type, high variability is generally reported in ferentiation), the morphological features of americanus) hemocytes: Classification, the literature for differing crustacean species. hyalinocytes from C. aestuarii (a central nucle- differential counts, and associated agglu- In the present study, granule-containing cells us, a high nucleus:cytoplasm ratio, the tinin activity. J Invertebr Pathol 1978;31: were the most abundant cell type. Conversely, absence of evident granules in cytoplasm) 194-203. in M. rosenbergii, hyalinocytes comprised 70% resembles those of undifferentiated stem cells. 10. Stang-Voss C. Ultrastructure of inverte- of total haemocytes and no semigranulocytes This aspect will be better evaluated in future brate hemocytes. V. Hemocytes of Astacus were found.30 In Sicyonia ingentis, 50-60% of studies by means of specific antibodies, such astacus (L.) (Crustacea). Z Zellforsch the circulating haemocytes were hyalino- as anti-CD34 previously used to detect stem Mikrosk Anat 1971;122:68-75. cytes,31 whereas semigranulocytes and granu- cells in bivalves.34 11. Toney ME Jr. Morphology of the blood cells locytes were 30% and 10% of the total, respec- Summarising, light microscopy and cyto- of some Crustacea. Growth 19585;22:35- tively. High percentages of hyalinocytes (5-8 chemical analyses performed in the present 50. times more abundant than granulocytes) were study allowed us to identify three main haemo- 12. Bodammer JE. Cytological observations on also observed in the crab Eriocheir sinensis,32 cyte types in haemolymph from the crab C. aes- the blood and hemopoietic tissue in the in the lobster Panulirus interruptus (about tuarii. Although preliminary morphological crab, Callinectes sapidus. Cell Tissue Res 56%)7 and in the crayfish Procambarus clarki investigation of haemocytes under light micro- 1978;187:79-86. (more than 70%).33 Conversely, in the lobster scope is considered necessary by investigators, 13. Smith VJ, Ratcliffe NA. Host defense reac- H. americanus and the crab Loxorhynchus ultrastructural and functional studies are also tions of the shore crab, Carcinus maenas, grandis, semigranulocytes reached more than needed to better distinguish crab cell types and in vitro. J Mar Biol Ass UK 1978;58:367-79. 60% of the total cell number.7 Although the sig- clarify their role in immune responses. To this 14. Ravindranath MH. The hemocytes of an nificance of this marked variability in the rela- aim, future studies will be performed. isopod Ligia exotica roux. J Morphol 1974; tive proportions of each haemocyte type among 144:11-22. crustacean species remains unclear, influ- 15. Johnston MA, Elder HY, Davies PS. ences of moult cycle, diet, harvesting, diseases Cytology of Carcinus haemocytes and their and environmental contaminants cannot be function in carbohydrate metabolism. excluded. References Comp Biochem Physiol 1973;46A:569-74. All haemocytes types from C. aestuarii were 16. William AJ, Lutz PL. Blood cell types in PAS positive, indicating that they contained 1. Bauchau AG. . In: Ratcliffe NA, Carcinus maenas and their physiological carbohydrates. However, carbohydrate distri- Rowley AF (eds). Invertebrate Blood Cells, role. J Mar Biol Ass UK 1975;55:671-4. bution differed among cell types (see Results). Vol 2. London: Academic Press 1981; pp 17. Manjula PL, Rahman K, Abraham TJ. Interestingly, a similar carbohydrate distribu- 385-420. Haemocyte classification and differential tion was previously described by Gargioni and 2. Johansson MW, Keyser P, Sritunya- counts in the Indian spiny lobster, Barracco8 for haemocytes from P. paulensis, M. lucksana K, Söderhäll K. Crustacean Panulirus homarus (linneaus). J Aquacult acanthurus and M. rosenbergii. Conversely, haemocyte and haemato-poiesis. Aquacul- Tropics 1997;12:113-21. William and Lutz16 classified C. maenas haemo- ture 2000;191: 45-52. 18. Clare AS, Lumb G. Identification of haemo- cytes in two types, one with glycogen-contain- 3. Ghiretti-Magaldi A, Milanesi C, Tognon G. cytes and their role in clothing in the blue ing granules and the other with non-glycogen- Hemopoiesis in Crustacea Decapoda: ori- crab Callinectes sapidus. Mar Biol 1994; containing granules. Overall, the results of the gin and evolution of hemocytes and 118: 601-10. present study confirm the important role of cyanocytes of Carcinus maenas. Cell Differ 19. Battison A, Cawthorn R, Horney B. crab haemocytes in both synthesis and trans- 1977;6:167-86. Classification of Homarus americanus port of carbohydrates. According to the hypoth- 4. Martin GG, Hose JE, Choi M, et al. hemocytes and the use of differential esis of Bauchau,1 we suggested that haemo- Organization of hematopoietic tissue in hemocyte counts in lobsters infected with cytes from C. aestuarii can regulate the intermolt lobster, Homarus ameri- Aerococcus viridans var. homari haemolymph sugar levels, depending on need. canus. J Morphol 1993;216:65-78. (Gaffkemia). J Invertebr Pathol 2003;84: Sudan Black B staining demonstrated that 5. Chaga O, Lignell M, Söderhäll K. The 177-97. all haemocyte types of C. aestuarii contained haemopoietic cells of the freshwater cray- 20. 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