Morphological, Cytochemical and Ultrastructural Aspects of Blood Cells in Freshwater Stingray Species in the Middle Rio Negro Basin, Amazonian, Brazil

Examined the morphology, dimensions, cytochemical staining reactions and ultrastructure of blood cells from three freshwater stingray species, Potamotrygon wallacei, Potamotrygon motoro and Paratrygon aiereba, living in the waters of the middle Rio Negro basin (Barcelos, Amazonas, Brazil). We identied erythrocytes, erythroblasts, thrombocytes and four types of leukocyte (basophils, heterophils, lymphocytes and monocytes) in the blood of these stingray species. In all the freshwater stingrays studied, the shape and dimensions of these cells were similar to those of marine elasmobranchs. A positive PAS reaction occurred in heterophils and thrombocytes, and a weak reaction in lymphocytes and monocytes, while a metachromasia reaction only occurred in basophils. Sudan black staining was positive for thrombocytes and lymphocytes, and only a weak reaction occurred in heterophils. Basophils and heterophils were the only cells stained with bromophenol blue, while no peroxidase reaction was observed in any leukocyte type. This is the rst study to establish the dimensions and cytochemical staining reactions of blood cells in Amazonian stingray species. Since these elasmobranch species are exported as ornamental sh to countries worldwide, this study can contribute towards establishing standards for blood constituents that may be helpful in assessing the health and welfare of these sh in articial systems.


Introduction
The family Potamotrygonidae is a unique elasmobranch group composed of freshwater stingray species distributed along most of the great uvial systems of South America ending at the Atlantic Ocean or Caribbean Sea (Compagno and Cook 1995;Lovejoy 1996). These Neotropical freshwater stingrays are currently classi ed into four valid genera: Plesiotrygon, Paratrygon, Potamotrygon (Carvalho et al. 2003) and, in a recent contribution (Carvalho and Lovejoy 2011), the new Heliotrygon genus, with two species, H. gomesi and H. rosae. However, great effort and research investment are needed in order to achieve better understanding of the diversity and taxonomic status of this family (Rosa et al. 2010).
Freshwater stingrays are an important component of Amazonian biodiversity. They have great socioeconomic importance, especially because of their use in the international ornamental sh trade, and because they represent an alternative source of income for riverine communities living along the tributaries of the middle Rio Negro basin (Chao et al. 2001). There is a relationship of freshwater stingrays with shermen, especially by the presence of stingers that can cause accidents (Oliveira et al. 2015). Four valid species are found in this black water system: Potamotrygon motoro (Müller & Henle 1841), Potamotrygon orbignyi (Castelnau 1855), Potamotrygon schroederi (Fernández-Yépez 1958) and Paratrygon aiereba (Müller & Henle 1841). In addition, a new species known as Potamotrygon wallacei (cururu stingray) (Carvalho et al. 2016), is currently being identi ed and scienti cally described. This species is probably endemic to this region, with a hotspot concentrated in the Mariuá archipelago, near the municipality of Barcelos (Amazonas, Brazil).
Investigations on the blood constituents of elasmobranch sh have been conducted on several marine species, especially sharks (Valenzuela et al. 2003;Luer et al. 2004 This study aimed to investigate the morphology, dimensions, cytochemical staining reactions and ultrastructure of blood cells from three freshwater stingray species, P. wallacei (cururu stingray), P. motoro and P. aiereba, living in the black waters of the middle Rio Negro basin (Barcelos, Amazonas, Brazil). Since Brazil and other Amazonian countries export these species as ornamental sh to consumers around the world, these results will contribute towards establishing standards for blood constituents that may be helpful in assessing the health and welfare of these sh in arti cial systems, especially in relation to the ornamental sh trade.

Material And Methods
Study area and specimen collection Specimens of the Amazonian stingrays Potamotrygonwallacei (cururu stingray; n= 53), Potamotrygon motoro (n= 55) and Paratrygon aiereba (n= 32) were collected from the Mariuá archipelago (Collection Licence: 15116-1 IBAMA). This is the largest complex of islands that exists in continental waters (more than 700 islands; IBGE 2012), and it is located in the black waters of the middle Rio Negro basin, near the municipality of Barcelos (Amazonas, Brazil). These sh were caught at different sites within the archipelago, including beaches, lakes, small streams (igarapés), and areas of ooded forest (igapós), between January 2006 and October 2011. They were all caught by professional sherman at night (19:00 to 03:00), through active searching with the aid of a head ashlight, a paddle and a typical hand net (rapiché). We immediately anesthetized the captured stingrays with eugenol (0.2 g/L), and withdrew a blood sample (1.0-1.5 mL) from the gill arterial vessel (Tavares-Dias 2006) used the anticoagulant EDTA 10%. After these procedures, we measured the total length (TL, cm), disc width (DW, cm) and body weight (BW, kg) of each specimen. All the stingrays sampled recovered from the anesthetic and were safely returned to their respective capture site.
For cytochemical staining and ultrastructure examination of different blood cell types, ten individuals of P. wallacei, P. motoro and P. aiereba were caught near the Daracuá community, within the Mariuá archipelago, by professional sherman. These stingrays were transported by boat (journey of 24 hours) to the Laboratory for Physiology Applied to Aquaculture (LAFAP), at the National Amazon Research Institute (Instituto Nacional de Pesquisas da Amazônia, INPA) in Manaus (Amazonas, Brazil). At the laboratory, they were acclimatized in 5000-liter tanks for 48 hours, with constant water changes and oxygenation so that the sh would recover from the stress that resulted from the capture and transportation procedures. After this period, blood sample (1.0 mL) was collected from the gill arterial vessel used the anticoagulant EDTA 10% (Oliveira et al. 2015c). Immediately after blood collection, blood smears were made. They were then determined the biometric parameters (TL, DW and BW).

Morphological blood cells and morphometric measurements
For this experimental procedure, we took fresh blood samples from P.wallacei (n = 43), P. motoro (n = 45) and P. aiereba (n= 32). We stained these blood smears with a combination of May-Grünwald-Giemsa-Wright in order to identify cells and make morphometric measurements (µm) 100 samples, with the aid of an optical microscope and a millimeter ruler.

Cytochemical staining reactions
For this experimental procedure, we took fresh blood samples from 10 specimens of each stingray species for smear preparation. The presence and intensity of glycogen deposits inside blood cells was con rmed by using the periodic acid-Schiff (PAS) method. Controls for this reaction were obtained through smears exposed to salivary amylase digestion for 60 minutes.
The peroxidase reaction was carried out by using the ortho-toluidine method in the presence or absence of hydrogen peroxide. The reactions products were subjected to nuclear staining using Harris hematoxylin (Tavares-Dias and Moraes 2006).
Reactions for metachromasia were tested in blood smears that were xed in 1% lead subacetate for 10 minutes and subsequently stained with 0.2% toluidine blue for 50 minutes (Tavares-Dias and Moraes 2006). The presence of lipids in different blood cell types was con rmed in blood smears that had previously been xed with 70% ethanol for 5 seconds and then were stained with 0.3% Sudan Black B solution.
In order to identify total protein, blood smears were xed in formalin, stained with bromophenol blue for 15 minutes and then immersed in 0.5% acetic acid, washed in phosphate buffer and nally dehydrated in butyl alcohol. Reticulocytes were identi ed using a solution of brilliant cresyl blue and blood (1:1), which was homogenized, kept in a water bath for 20 minutes at 37 °C and stained with a combination of May-Grünwald-Giemsa-Wright (Tavares-Dias and Moraes 2006). The results from the cytochemical staining were expressed qualitatively, according to the intensity of reactions observed for each blood cell type, i.e. negative reaction (-), weak positive reaction (+) and positive reaction (++).

Ultrastructural analysis
The blood cell types were characterized ultrastructurally in four out of the ten individuals of each stingray species that had been acclimatized for cytochemical studies. Blood samples were taken from the gill vessel (Oliveira et al. 2012), and were centrifuged at 750 g for 15 min to obtain pellets containing erythrocytes, thrombocytes and leukocytes. We immediately xed these pellets in 0.1 M sodium cacodylate solution (pH 7.4) containing 2.5% glutaraldehyde and 2.0% paraformaldehyde, at 4 °C for 2.5 hours. We then immersed these samples in a 0.2 M sodium cacodylate solution (pH 7.4) containing 1% osmium tetroxide, at 4 °C for one hour. After these procedures, the samples were dehydrated and embedded in Araldite resin (Sigma-Aldrich, USA) and sections were cut using a Reichert OM-U3 ultratome,

Results
The mean values for total length, disc width and body mass of the specimens of P. wallacei, P. aiereba and P. motoro are shown in Table 1.

Morphological blood cells and morphometric measurements
The blood smears from P. wallacei, P. motoro and P. aiereba revealed erythroblasts, mature erythrocytes, thrombocytes, lymphocytes, monocytes, heterophils and basophils, of similar sizes among such species.
Monocytes were the largest cells in these three elasmobranch species, in comparison with the other leukocyte cells ( Table 2).
The mature erythrocytes were very similar in shape and size in these three Amazonian stingray species. Under an optical microscope, they presented as elliptical cells with abundant hyaline cytoplasm and a nucleus that was usually centered and condensed, and its shape followed that of the cell (Figure 1-I). The erythroblasts were rounded cells and easily differentiated from mature erythrocytes by their pale or hyaline cytoplasm and a higher proportion of nucleus in relation to cytoplasm (N:C ratio) (Figure 1-I).
The lymphocytes presented different sizes and irregular shapes, which were mostly elliptical and rarely oval, and with a nucleus occupying a large part of the basophilic cytoplasm. They presented cytoplasmic projections without visible granulations, and sometimes presented vacuoles (Figure 1-II). The thrombocytes were generally fusiform, with hyaline cytoplasm, their nucleus occupied almost the entire cell and its shape follows that of the cell (Figure 1-III). The monocytes were predominantly oval, with nucleus similar thrombocytes (Figure 1-III). The heterophils were predominantly oval, with large amount of heterophilic coarse granules and a nucleus that was generally eccentric (Figure 1-IV). The basophils were also predominantly oval, with basophilic granules and a nucleus that was eccentric and generally bilobulated (Figure 1-V).

Cytochemical staining reactions
The thrombocytes and leukocytes did not show any differences in cytochemical reactions between the three species of rays (Table 3) (Figure 4-II). Presence of reticulocytes was observed in erythrocytes, thus indicating the presence of crosslinking material fragments that did not stain with traditional dyes. There was no positive peroxidase reaction, although the metachromasia reaction was found (Figure 4-III). This was characterized by use of a blue reagent, which reacted with the red-colored blood of these freshwater rays.

Ultrastructural analysis
The thrombocytes were generally round and spindle-shaped. In their cytoplasm, a canalicular system with various sizes of vesicles of different sizes and canaliculi was occasionally found, along with glycogen pellets, granules and numerous mitochondria ( Figure 5-I). Lymphocytes presented amorphous forms, with sparse cytoplasm. Presence of vacuoles and few mitochondria was observed, and the nucleus occupied almost the entire cell, with dense chromatin in the periphery and no evident nucleolus ( Figure 5-II). The monocytes presented nuclei with peripheral heterochromatin and cytoplasm with mitochondria, secretion vesicles, secretion granules and endoplasmic reticulum. Because of scarcity of basophils in the blood, this type of granulocyte could not be found in these potamotrygonids in this study. Staining of heterophils revealed the presence of heterochromatin, and there were large numbers of granules that might have been glycogen, lipids and proteins, but could not be distinguished. 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.

Discussion
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, quanti cation of the number of circulating reticulocytes can provide information about erythropoietic activity, and therefore about animal health status. 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 intraspeci c differences relating to the environment.
Thrombocytes in elasmobranchs are blood cells with functions analogous to mammals' platelets, which play a role in homeostasis Walsh and Luer 2004). In dog sh (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 , 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 ngerlike 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 identi cation , thereby contributing towards the confusing terminology of elasmobranch leukocytes , 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  ). The size of the lymphocytes of these Amazonian rays was slightly smaller than those of the shark C. coelolepis (Sherburne 1973 Old and Huveneers 2006). Granulocytes have been reported in several elasmobranch species, but they are di cult 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 (Gri th et al. 1973). Identi cation of neutrophils and eosinophils in these potamotrygonids can be correlated with the extreme di culty of the methods for staining smears and/or with incorrect classi cation 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 (dog sh shark) (Sherburne 1973 It was reported the existence of neutrophils and eosinophils in the blood of an individual of P. motoro and mentioned that di culty 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 However, the present study was the rst 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 ).
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 Veiga et al. 2000).
Since basophils are rare leukocytes in the blood of P. wallacei, P. motoro and P. aiereba, their existence could be con rmed 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 ndings 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 sh, which are extremely important for the aquarium market, can be quanti ed. 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.

Declarations
Authors contributions ATO and JLM conceived the study. ATO, JRGL, MQC, MLG and JLM designed the study. ATO, JRGL, MQC and MTD undertook laboratorial analyses. JPL, PHRA and MTD drafted the paper with contributions from all other authors. All authors read and approved the nal manuscript.

Data availability
The datasets in this study are available from the corresponding author on reasonable request.

Consent for publication
Not applicable.