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

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 \ub5m in diameter and 30-50 fl in volume, the second was of about 6-12 \ub5m in diameter and over 200 fl in volume. Mean cell diameter and volume were 8.20 \ub1 1.7 \ub5m and 272.30 \ub1 143.5 fl, respectively. Haemocytes observed under light microscope were distinguished in three cell types: granulocytes (28%; 11.94 \ub1 1.43 \ub5m in diameter) with evident cytoplasmic granules, semigranulocytes (27%; 12.38 \ub1 1.76 \ub5m in diameter) with less granules than granulocytes, and hyalinocytes (44%; 7.88 \ub1 1.6 \ub5m in diameter) without granules. In addition, a peculiar cell type was occasionally found (about 1%): it was 25-30 \ub5m 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\u2019s dye confirmed that granulocytes and semigranulocytes were larger than hyalinocytes. Pappenheim\u2019s panoptical staining and Ehrlich\u2019s 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