Most vertebrates have seven blood cell types: erythrocytes, thrombocytes, lymphocytes, eosinophils, basophils, monocytes and neutrophils (Tavares-Dias and Moraes 2003; Canfield 1998). The morphology of each cell type appears to be similar, except for neutrophils. Which in some cases are replaced by heterophils, which present the same immunological function (Canfield 1998; Davis et al. 2008; Hawkey and Dennett 1989). It was reported that erythrocytes, thrombocytes, lymphocytes, monocytes, neutrophils and eosinophils were present in freshwater potamotrygonids (Griffith et al. 1973).
In contrast, no presence of eosinophils was observed in blood from Amazon stingrays, thus suggesting that heterophils have some importance in the immune defense of these potamotrygonids.
In the potamotrygonids of this study, reticulocytes were revealed through the presence of ribonucleoproteins inside some erythrocytes. High amounts of ribonucleoproteins indicate premature release of erythrocytes into the bloodstream (Tavares-Dias and Moraes 2006). Therefore, quantification of the number of circulating reticulocytes can provide information about erythropoietic activity, and therefore about animal health status.
Erythrocytes are generally larger in lower orders and variations in size may occur within species of the same order (Canfield 1998). The erythrocytes of freshwater stingrays were smaller than of those of sharks Centroscymnus coelolepis (Barbosa du Bocage and de Brito Capello 1864) (Sherburne 1973), and about two times larger than in freshwater and marine teleosts (Vázquesz and Guerrero 2007) and Dicentrarchus labrax L.. (Esteban et al. 2000) The morphological features of erythrocytes of freshwater stingrays are similar to those of marine elasmobranchs such as the rays Dasyatis sabina (Lesueur 1824), Raja eglanteria (Bosc 1800) (Luer et al. 2004; Walsh and Luer 2004), Raja microocellata (Montagu 1818), Raja brachyura (Lafont 1871) and Raja sp. (Aragort et al. 2005), and the sharks Squalus acanthias (Linnaeus 1758) (Clewley et al. 2002), Schroederichthyes chilensis (Guichenot 1848) (Valenzuela et al. 2003), Ginglymostoma cirratum (Bonnaterre 1788) and Carcharhinus limbatus (Müller & Henle 1839).
In addition, in the shark C. coelolepis, immature erythrocytes (erythroblasts) may be smaller than mature erythrocytes (Sherburne 1973), and this characteristic was also found in these potamotrygonid stingrays. Therefore, these results do not show intraspecific differences relating to the environment.
Thrombocytes in elasmobranchs are blood cells with functions analogous to mammals’ platelets, which play a role in homeostasis (Luer et al. 2004; Walsh and Luer 2004). In dogfish (S. canícula), it was demonstrated that blood thrombocytes remove antigenic substances, such as colloidal charcoal particles (Morrow and Pulsford 1980). The cell sizes and morphological characteristics of the thrombocytes of freshwater stingrays were similar to those reported in the sharks S. chilensis (Valenzuela et al. 2003) and C. leucas (Luer et al. 2004), and different from C. plumbeus, which presented cytoplasmic granules (Arnold 2005). Moreover, in the blood of the shark C. coelolepis, the form known as "drop" (with fingerlike cytoplasmic projection), was observed (Sherburne 1973), but this was not found in the Amazonian stingrays of this study.
In blood smears from marine elasmobranchs, leukocytes at different stages of maturation are frequently observed. This can cause incorrect identification (Luer et al. 2004), thereby contributing towards the confusing terminology of elasmobranch leukocytes (Luer et al. 2004), and also causing errors in identifying small monocytes and large lymphocytes (DaMatta et al. 2009). In the present study, lymphocytes presented shapes ranging from round to amorphous, and this has also been observed among lymphocytes in C. coelolepis (Sherburne 1973), S. chilensis (Valenzuela et al. 2003), G. cirratum (Valenzuela et al. 2003; Luer et al. 2004), C. plumbeus (Arnold 2005), R. microocellata, R. brachyura, R. sp. (Aragort et al. 2005), O. maculatus, O. ornatus, O. sp.(Old and Huveneers 2006) and R. typus (Dove et al. 2010). The size of the lymphocytes of these Amazonian rays was slightly smaller than those of the shark C. coelolepis (Sherburne 1973).
The morphological characteristics of this cell type were similar to those observed in other elasmobranchs (Valenzuela et al. 2003; Luer et al. 2004; Arnold 2005; Dove et al. 2010; Aragort et al. 2005; Old and Huveneers 2006). Granulocytes have been reported in several elasmobranch species, but they are difficult to identify and classify because of the great variations in shape and size and the poor staining of the cells (Valenzuela et al. 2003). In the present study, in blood of freshwater stingrays, two types of granulocytes have been showed: heterophils and basophils. It was reported that the most common granulocytes in the blood of elasmobranchs were heterophils, while basophils were rare in blood (Valenzuela et al. 2003). It was shown that neutrophils and eosinophils were present in the blood of potamotrygonids (Griffith et al. 1973). Identification of neutrophils and eosinophils in these potamotrygonids can be correlated with the extreme difficulty of the methods for staining smears and/or with incorrect classification of the different types of leukocyte. Presence of heterophils and basophils with the same morphological features as in these Amazonian stingrays was observed in C. coelolepis (dogfish shark) (Sherburne 1973), S. chilensis (catshark) (Valenzuela et al. 2003), C. limbatus (blacktip shark) (Luer et al. 2004) and R. typus (whale shark) (Dove et al. 2010).
It was reported the existence of neutrophils and eosinophils in the blood of an individual of P. motoro and mentioned that difficulty in distinguishing neutrophils from heterophils had been found (Oliveira et al. 2015b). In the present study, no neutrophils were found. Instead, there were heterophilic granulocytes with morphological features distinct from neutrophils. However, these had heterophilic functions resembling phagocytosis, as also seen among neutrophils, as indicated by the presence of glycogen, lipids and proteins in P. wallacei, P. motoro and P. aiereba. Glycogen is an important source of cellular energy reserves for the innate defense mechanisms that occur, especially during the process of phagocytosis (Tavares-Dias and Moraes 2006; Ueda et al. 2001).
In the class Chondrichthyes, it was studied the cytochemical characteristics of leukocyte chimeras in the species Callorhynchus milii (Bory de Saint-Vincent 1823), Chimaera phantasma (Jordan & Snyder 1900), Hydrolagus novaezealandiae (Fowler 1911), Hydrolagus sp., Harriotta raleighana (Goode & Bean 1895) and Rhinochimaera pacifica (Mitsukuri 1895) (Hine and Wain 1988). They reported that the enzyme esterase in the subclass Holocephali was very different from this enzyme found in elasmobranchs. However, the present study was the first aimed at determining the functions of blood cell types in potamotrygonid species. A positive PAS reaction was observed in thrombocytes of P. wallacei, P. motoro and P. aiereba, but the reaction in lymphocytes and monocytes was weak. Thrombocytes are cells that act on blood coagulation (Hayhoe et al. 1994), but they also play an important role in the immune activity of elasmobranchs (Luer et al. 2004).
There was no peroxidase reaction in any of the blood cells of P. wallacei, P. motoro and P. aiereba. Peroxidase is an important lysosomal enzyme involved in intracellular digestion, and one of its main features is that it marks absence of eosinophilic and neutrophilic granulocytes in the species investigated here. However, this lack of peroxidase may be accompanied by compensatory development of other antibacterial components, such as cationic proteins (Luer et al. 2004; Veiga et al. 2000).
Since basophils are rare leukocytes in the blood of P. wallacei, P. motoro and P. aiereba, their existence could be confirmed through the metachromasia reaction. In addition, these potamotrygonids demonstrated presence of lipids in thrombocytes and lymphocytes, but to a lesser degree than in heterophils. Similarly, in Xiphophorus helleri (Heckel 1848), a Sudan black reaction was also demonstrated in monocytes and lymphocytes (Schutt et al. 1997). However, in other teleosts, this reaction has been described in neutrophil granules (Tavares-Dias 2006). Phagocytic leukocytes can use lipids as an energy source, thereby degrading these constituents through the action of cytoplasmic enzymes.
The proteins in leukocyte granules are involved in host defense and microorganism death (Tavares-Dias 2006). The heterophils and basophils of P. wallacei, P. motoro and P. aiereba were positive for staining with bromophenol blue, similarly to what had previously been found in eosinophils from S. brasiliensis (Tavares-Dias 2006) in Amazonian turtles (Oliveira et al. 2011). It was observed a positive reaction in basophils, eosinophils and neutrophils from P. motoro (Oliveira et al. 2015b). Therefore, these results indicate that these proteins play an important role in the innate defense of animals, which is possibly performed by these granulocytes.
The ultrastructural analyses on leukocytes from P. wallacei, P. motoro and P. aiereba were similar to each other and comparable with the findings from the sharks G. cirratum (Hyder et al. 1983) and S. canicular (Morrow and Pulsford 1980). The morphology and sizes of the different cell types were similar to those of marine rays and sharks. It is very important to characterize the types of leukocytes in rays in order to provide basic knowledge of these cells and to make correlations with health conditions. In this manner, the cell types of these fish, which are extremely important for the aquarium market, can be quantified. The cytochemical characteristics of the heterophils indicated that these major granulocytes were important in the immune defense of Amazonian potamotrygonids. The blood cell features of wild native stingrays may be useful for making diagnoses and comparisons among these same species under farmed conditions.