First cytochemical study of haemocytes from the crab Carcinus aestuarii (Crustacea, Decapoda)

Abstract

For the first time, a morphological study of haemocytes from the crab Carcinus aestuarii was carried out by means of light microscopy and differing cytochemical assays. Analysis of haemocyte size frequency distribution (performed by means of a Coulter Counter) revealed the presence of two distinct haemocyte fractions in C. aestuarii haemolymph, depending on cell size. The first fraction was of about 3-5 microm in diameter and 30-50 fL in volume, the second was of about 6-12 microm in diameter and over 200 fL in volume. Mean cell diameter and volume were 8.20+/-1.7 microm and 272.30+/-143.5 fL, respectively. Haemocytes observed under light microscope were distinguished in three cell types: granulocytes (28%; 11.94+/-1.43 microm in diameter) with evident cytoplasmic granules, semigranulocytes (27%; 12.38+/-1.76 microm in diameter) with less granules than granulocytes, and hyalinocytes (44%; 7.88+/-1.6 microm in diameter) without granules. In addition, a peculiar cell type was occasionally found (about 1%): it was 25-30 microm in diameter and had a great vacuole and a peripheral cytoplasm with granules. Granulocyte and semigranulocyte granules stained in vivo with Neutral Red, indicating that they were lysosomes. Giemsa's dye confirmed that granulocytes and semigranulocytes were larger than hyalinocytes. Pappenheim's panoptical staining and Ehrlich's triacid mixture allowed to distinguish granule-containing cells (including semigranulocytes) in acidophils (64%), basophils (35%) and neutrophils (1%). Hyalinocytes showed always a basophilic cytoplasm. Haemocytes were positive to the PAS reaction for carbohydrates, even if cytoplasm carbohydrate distribution varied among cell types. Lastly, lipids were found on cell membrane and in cytoplasm of all haemocyte types in the form of black spots produced after Sudan Black B staining. The morphological characterisation of C. aestuarii haemocytes by light microscopy was necessary before performing both ultrastructural and functional studies of circulating cells.

Figure 1

An example of haemocyte size frequency distribution in C. aestuarii. Haemocyte diameter (A), expressed in µm, and haemocyte volume (B), expressed in femtolitres (fL).

Figure 2

Unfixed haemocytes of C. aestuarii, observed in vivo (A–D). g: granulocyte; sg: semigranulocyte; h: hyalinocytes; lp: lipoprotein cell. Bar length: 5 µm.

Figure 3

C. aestuarii haemocytes stained with Giemsa's dye (A–D) and vitally stained with Neutral Red, showing lysosomes (arrows) (E, F). g: granulocytes; sg: semigranulocytes; h: hyalinocytes; lp: lipoprotein cells. Bar length: 5 µm

Figure 4

C. aestuarii haemocytes stained with Pappenheim’s (A–D) and Ehrlich’s triacid mixtures (E, F); h: hyalinocytes; Ag: acidophil granulocytes; Bg: basophil granulocytes; Ng: neutrophil granulocytes. Bar length: 5 µm.

Figure 5

C. aestuarii haemocytes stained with PAS (A–C) and Sudan Black B (D–F). Red arrows indicate black deposits on cell membrane and in cytoplasm; g: granulocytes; sg: semigranulocytes; h: hyalinocytes. Bar length: 5 µm.