View metadata, citation and similar papers at core.ac.uk brought to you by CORE European Journal of Histochemistry 2010; volume 54:e9 provided by PubMed Central First cytochemical study of Introduction Correspondence: Valerio Matozzo, Department of haemocytes from the crab Biology, University of Padova, via Ugo Bassi, Carcinus aestuarii (Crustacea, Like other arthropods, Crustacea have an 58/B, 35131 Padova, Italy. Decapoda) open vascular system in which numerous Tel.: +39.049.8276201 - Fax: +39.049.8276199. haemocytes freely circulate in haemolymph. E-mail: [email protected] Crustacean 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- Carcinus maenas, 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 Animals 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 crabs 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).