Cytometric and morphological characterization of hemocytes from four species of freshwater decapod crustaceans

Characterization and classification of invertebrate hemocytes is a powerful tool for determining the physiological effect of environmental stress. Hemocytes from four decapod crustacean species Dilocarcinus pagei, Zilchiopsis collastinensis, Aegla uruguayana and Macrobrachium borellii were analyzed by flow cytometry followed by morphological characterization by optical microscopy. Based on morphological cell characteristics, three categories of hemocytes were identified: granulocytes with abundant granularity accounting for 1.87% (D. pagei), 5.23% (Z. collastinensis), 0.98% (A. uruguayana) and 2.41% (M. borellii), semigranulocytes with lower granularity accounting for 2.22% (D. pagei), 4.81% (Z. collastinensis), 2.38% (A. uruguayana) and 68.19% (M. borellii) and, finally, hyalinocytes with almost no granularity accounting for 89.87% (D. pagei), 65.75% (Z. collastinensis), 92.33% (A. uruguayana) and 22.04% (M. borellii). Analysis of the total hemocyte count (cells/ml) showed the following results: 4.3 ± 0.58 (x106) in D. pagei, 2.3 ± 0.33 (x106) in Z. collastinensis; 3.1 ± 0.56 (x106) in A. uruguayana and 2.6 ± 0.25 (x106) in M. borellii. This study demonstrates the applicability of flow cytometry to effectively differentiate and enumerate circulating hemocytes in native crustacean species, providing novel insights into the use of this method for assessing physiological status in these species. In addition, interspecific variability in these cellular parameters was observed. The results contribute to the understanding of the innate immune system in these four crustacean species and allow establishing a baseline to identify welfare or stress conditions in these animals.