Morphological, cytochemical and ultrastructural aspects of blood cells in freshwater stingray species in the middle Rio Negro basin of Amazonian Brazil

In the present work, we 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 identified erythrocytes, erythroblasts, thrombocytes and four types of leukocytes (basophils, heterophils, lymphocytes and monocytes) in the blood of these stingray species. In all the freshwater stingray species studied, the shapes and dimensions of these cells were similar to those of marine elasmobranchs. Positive PAS staining occurred in heterophils and thrombocytes, and weak staining occurred in lymphocytes and monocytes, while metachromasia only occurred in basophils. Positive Sudan Black B staining was observed in thrombocytes and lymphocytes, and weak staining occurred in heterophils. Basophils and heterophils were the only cells with positive bromophenol blue staining, while no peroxidase staining was observed in any of the four leukocyte types. This is the first study to establish the dimensions and cytochemical staining profiles of blood cells in Amazonian stingray species. Because these elasmobranch species are exported as ornamental fish to countries worldwide, this study can contribute to establishing standards for blood constituents that may be helpful in assessing the health and welfare of these fish in artificial systems.

Hematological evaluations are becoming routine practice for assessing the health of fish and other animals 19,21,[24][25][26][27][28][29][30][31][32][33] . Studies on blood leukocytes can reveal the characteristics of the immune systems of different fish species 34,35 , including free-living Amazonian stingrays. Hematological investigations have relied on classical Romanowsky staining with the Leishman, Wright, May, Grünwald and Giemsa used to identify leukocytes [36][37][38] , but cell-based classifications of stingray leukocyte cells are not always reliable using classical staining methods because the staining procedures vary, which can lead to errors in the identification of a cell type. Thus, cytochemical staining of leukocytes in blood may be particularly useful for identifying cell lineages and may indicate cell function.
This study aimed to investigate the morphology, dimensions, cytochemical staining reactivity 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). Because Brazil and other Amazonian countries export these species as ornamental fish to consumers around the world, these results will contribute to establishing standards for blood constituents that may be helpful in assessing the health and welfare of these fish in artificial systems, especially in relation to the ornamental fish trade.

Materials and methods
Study area and specimen collection. Specimens of the Amazonian stingrays Potamotrygon wallacei (cururu stingray; n = 53), Potamotrygon motoro (n = 55) and Paratrygon aiereba (n = 32) were collected from the Mariuá archipelago (license: 15116-1 IBAMA). This is the largest complex of islands that exists in continental waters (more than 700 islands), and it is located in the black waters of the middle Rio Negro basin near the municipality of Barcelos (Amazonas, Brazil). The fish were caught at different sites within the archipelago, including beaches, lakes, small streams (igarapés), and areas of flooded forest (igapós), between January 2006 and October 2019. Professional fisherman caught the fish at night (19:00 to 03:00) through active searching with the aid of a head flashlight, paddle and typical hand net (rapiché). We immediately anesthetized the captured stingrays with eugenol (0.2 g L −1 ) and withdrew a blood sample (1.0-1.5 mL) from the gill arterial vessel 18 using 10% EDTA as an anticoagulant 20 . 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 sites.
For cytochemical staining and ultrastructural examination of different blood cell types, ten P. wallacei, P. motoro and P. aiereba stingrays were caught by professional fishermen near the Daracuá community within the Mariuá archipelago. These stingrays were transported by boat (journey of 24 h) 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, the stingrays were acclimatized in 5000-L tanks for 48 h, with constant water changes and oxygenation so that they would recover from the stress of being captured and transported. After this period, a blood sample (1.0 mL) was collected from the gill arterial vessel using 10% EDTA as an anticoagulant 20 . Then, the biometric parameters were determined (TL, DW and BW).

Morphological and morphometric measurements and quantification of blood cells. Fresh
blood samples were collected 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 stains to identify cells and make morphometric measurements (µm) of 100 samples of each cell type found (erythrocytes, leukocytes and thrombocytes), with the aid of an optical microscope and a millimeter ruler for determination of the largest and smallest cells.
Subsequently, the blood samples were used for leukocyte and total thrombocyte counts 37 and for differential leukocyte counts, which were based on the counts of 200 leukocytes 21 .
Cytochemical staining. We collected fresh blood samples from 10 specimens of each stingray species. The presence and intensity of glycogen deposits inside blood cells was confirmed by using the periodic acid-Schiff (PAS) method. Controls for this reaction were obtained through smears exposed to salivary amylase digestion for 60 min.
The peroxidase reaction was carried out by using the ortho-toluidine method in the presence or absence of hydrogen peroxide. The reaction products were subjected to nuclear staining using Harris hematoxylin 37 .
Reactions for metachromasia were tested in blood smears fixed in 1% lead subacetate for 10 min and subsequently stained with 0.2% toluidine blue for 50 min 37 . The presence of lipids in different blood cell types was confirmed in blood smears previously fixed with 70% ethanol for 5 s and then stained with 0.3% Sudan Black B solution 26  www.nature.com/scientificreports/ To identify total protein, blood smears were fixed in formalin, stained with bromophenol blue for 15 min, immersed in 0.5% acetic acid, washed in phosphate buffer and finally dehydrated in butyl alcohol. Reticulocytes were identified using a solution of brilliant cresyl blue and blood (1:1), which was homogenized, kept in a water bath for 20 min at 37 °C and stained with a combination of May-Grünwald-Giemsa-Wright stains 37 . The results from the cytochemical staining were expressed qualitatively, according to the intensity of staining observed for each blood cell type, i.e., negative staining (-), weakly positive staining (+) and positive staining (++).
Ultrastructural analysis. The blood cell types were characterized ultrastructurally in four of the ten stingrays from each species that had been acclimatized for cytochemical studies. Blood samples were taken from the gill vessel 18 and centrifuged at 750g for 15 min to obtain pellets containing erythrocytes, thrombocytes and leukocytes. We immediately fixed 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 h. 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, mounted on copper grids (200 mesh) and stained with 0.2% uranyl acetate solution and lead citrate solution for 15 min. The sections were analyzed using a transmission electron microscope at the Microscopy Center of the Institute of Biosciences at São Paulo State University (Universidade Estadual Paulista Julio de Mesquita Filho, UNESP) in Botucatu of São Paulo, Brazil.

Ethical approval. The study was conducted at the Department of Physiological Sciences, Federal University
Amazonas, in accordance with the Brazilian guidelines for animal experiments and was approved by the government of Amazonas (license: 15116-1), Brazil. All experiments were conducted according to local and ARRIVE guidelines 39 .

Research involving human participants and/or animals.
A total of 140 rays were captured in the natural environment, 130 of which were returned, and 10 were processed and registered in the collection.

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

Morphological and morphometric measurements and quantification of blood cells.
Blood smears from P. wallacei, P. motoro and P. aiereba revealed erythroblasts, mature erythrocytes, thrombocytes, lymphocytes, monocytes, heterophils and basophils of similar sizes among the species.
Mature erythrocytes were very similar in shape and size in the three Amazonian stingray species. Under an optical microscope, mature erythrocytes presented as elliptical cells with abundant hyaline in the cytoplasm, and the nucleus was usually centered and condensed with a shape that resembled the cell shape ( Fig. 1-I). Erythroblasts were rounded cells and were easily distinguished from mature erythrocytes by their pale appearance or by hyaline in the cytoplasm ( Fig. 1-I).
Lymphocytes presented different sizes and irregular shapes, which were mostly elliptical and rarely oval, with a nucleus occupying a large part of the basophilic cytoplasm. Lymphocytes presented cytoplasmic projections without visible granulations and sometimes presented vacuoles ( Fig. 1-II). Thrombocytes were generally fusiform, with hyaline in the cytoplasm; the nucleus occupied almost the entire cell and resembled the cell shape ( Fig. 1-III). Monocytes were predominantly oval, with a nucleus morphology similar to that of thrombocytes ( Fig. 1-III). Heterophils were predominantly oval, with a large amount of heterophilic coarse granules and a nucleus that was generally eccentric (Fig. 1-IV). Basophils were also predominantly oval, with basophilic granules and a nucleus that was eccentric and generally bilobulated ( Fig. 1-V).
In comparison with the other leukocyte cell types, monocytes were the largest cells in the three elasmobranch species (Table 2). Basophils and lymphocytes were the smallest cell types in the blood of the three freshwater stingray species investigated herein (Table 2).
Leukocyte and thrombocyte cell counts showed that lymphocytes and monocytes were the predominant blood cells, while heterophils and basophils were the least abundant blood cells in the three freshwater stingray species investigated (Table 3).
Cytochemical staining. Thrombocytes and leukocytes did not show any differences in cytochemical staining when comparing between the three stingray species (Table 4). Glycogen marking was observed in thrombo- www.nature.com/scientificreports/ cytes ( Fig. 2-I) and heterophils (Fig. 2-II), and there was weak positive staining in lymphocytes (Fig. 2-III) and monocytes ( Fig. 2-IV). Weak positive staining with Sudan black was observed in heterophils ( Fig. 3-I), lymphocytes (Fig. 3-II) and thrombocytes (Fig. 3-III). Positive identification of proteins using bromophenol blue occurred only in granules of heterophils (Fig. 4-I) and basophils (Fig. 4-II). The presence of reticulocytes was observed in erythrocytes, thus indicating the presence of crosslinking material fragments that were not stained with traditional dyes. There  www.nature.com/scientificreports/  Thrombocytes  www.nature.com/scientificreports/ was no positive peroxidase staining, although metachromasia was observed ( Fig. 4-III). This was characterized using a blue reagent that reacted with the red-colored blood of the freshwater stingrays.
Ultrastructural analysis. Thrombocytes were generally round and spindle-shaped. In the cytoplasm, a canalicular system with various-sized vesicles and canaliculi was occasionally observed, along with glycogen pellets, granules and numerous mitochondria (Fig. 5-I). Lymphocytes presented amorphous forms, with sparse cytoplasm. The 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 (Fig. 5-II). Monocytes presented nuclei with peripheral heterochromatin and cytoplasm with mitochondria, secretion vesicles, secretion granules and endoplasmic reticulum. Because basophils were scarce in the blood, they could not be found in the 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/or proteins, but they could not be distinguished.

Morphological and morphometric measurements and quantification of blood cells. Most ver-
tebrates have seven blood cell types: erythrocytes, thrombocytes, lymphocytes, eosinophils, basophils, monocytes and neutrophils 40,41 . The morphology of each cell type appears to be similar, except for neutrophils. In some cases, neutrophils are replaced by heterophils, which present the same immunological function 40,42,43 . It was reported that erythrocytes, thrombocytes, lymphocytes, monocytes, neutrophils and eosinophils were present in freshwater potamotrygonids 17 . In contrast, no eosinophils were observed in blood from Amazon stingrays, thus suggesting that heterophils have some importance in the immune defense of these potamotrygonids. In addition, there is a lack of standardization in the staining procedures adopted and in the classification of blood cell types. In the present study, erythroblasts, mature erythrocytes, thrombocytes, lymphocytes, monocytes, heterophils and basophils were observed.
In the potamotrygonids of this study, reticulocytes were identified by the presence of ribonucleoproteins inside some erythrocytes. High amounts of ribonucleoproteins indicate premature release of erythrocytes into the bloodstream 37 . Therefore, quantification of the number of circulating reticulocytes can provide information about erythropoietic activity and therefore about the animal health status.
In addition, in the C. coelolepis shark, immature erythrocytes (erythroblasts) may be smaller than mature erythrocytes 46 , and this characteristic was also observed in the three potamotrygonid stingray species.
Thrombocytes in elasmobranchs are blood cells with functions analogous to mammalian platelets, which play a role in homeostasis 13,16 . In dogfish (S. canicular), it was demonstrated that blood thrombocytes remove antigenic substances, such as colloidal charcoal particles 47 . The cell sizes and morphological characteristics of freshwater stingray thrombocytes were similar to those reported in the S. chilensis 12 and C. leucas 13 sharks and different from those of C. plumbeus, which presented cytoplasmic granules 11 . Moreover, in the blood of the C. coelolepis shark, the form known as "drop" (with fingerlike cytoplasmic projections) was observed 46 , but this was not detected in the Amazonian stingrays herein.
In blood smears from marine elasmobranchs, leukocytes at different stages of maturation are frequently observed. This can cause incorrect identification 13 , thereby contributing to the confusing terminology of www.nature.com/scientificreports/ elasmobranch leukocytes 13 and causing errors in identifying small monocytes and large lymphocytes 48 . In the present study, lymphocytes presented shapes ranging from round to amorphous, and this has also been observed among lymphocytes in C. coelolepis 46 , S. chilensis 12 , G. cirratum 12,13 , C. plumbeus 11 , R. microocellata, R. brachyura, R. sp. 44 , O. maculatus, O. ornatus, Orectolobus sp. 49 and R. typus 15 . The size of the lymphocytes in the three Amazonian stingray species was slightly smaller than that of the C. coelolepis shark 46 . The lymphocyte morphological characteristics were similar to those observed in other elasmobranchs [11][12][13]15,44,49 . 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 12 . In the present study, in the blood of freshwater stingrays, two types of granulocytes were detected: heterophils and basophils. It was reported that the most common granulocytes in the blood of elasmobranchs were heterophils, while basophils were rare in blood 12 . It was reported that neutrophils and eosinophils were present in the blood of potamotrygonids 17 . The identification of neutrophils and eosinophils in potamotrygonids can be correlated with the extreme difficulty of the methods for staining smears and/or with incorrect classification of the different types of leukocytes. Heterophils and basophils with the same morphological features observed in the three Amazonian stingrays were also observed in C. coelolepis (dogfish shark) 46 , S. chilensis (catshark) 12 , C. limbatus (blacktip shark) 13 and R. typus (whale shark) 15 .
Erythrocytes are generally larger in lower orders, and variations in size may occur within species of the same order 40 . The freshwater stingray erythrocytes were smaller than those of the Centroscymnus coelolepis shark (Barbosa du Bocage & de Brito Capello 1864) 46 and approximately two times larger than those in freshwater and marine teleosts 50 and in the Dicentrarchus labrax L. fish 51 .
The number of total leukocytes and thrombocytes observed in the present study was similar to the number reported for freshwater stingrays P. falkneri, P. motoro, P. orbignyi, and P. scobina 52 , in addition to P. schroederi and P. orbignyi 19 , as well as P. motoro, P. wallacei and P. aiereba 21 . For the differential leukocyte count, lymphocytes were the predominant cells. Oliveira et al. 20 and Oliveira et al. 21 also reported the same results; however, Brito et al. 52 reported that neutrophils and leukocytes were predominant.
Cytochemical staining. The existence of neutrophils and eosinophils in the blood of an individual P. motoro stingray was reported, and it was difficult to distinguish neutrophils from heterophils 19 . In the present study, no neutrophils were detected. Instead, there were heterophilic granulocytes with morphological features that were distinct from those of neutrophils. However, the granulocytes had heterophilic functions resembling phagocytosis, which is 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 phagocytosis 37,53 .
In the class Chondrichthyes, 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) 54 were studied. It was reported that the esterase enzyme in the Holocephali subclass was very different from that in elasmobranchs. However, the present study was the first aimed at determining the functions of blood cell types in potamotrygonid species. Positive PAS staining was observed in thrombocytes of P. wallacei, P. motoro and P. aiereba, but the staining in lymphocytes and monocytes was weak. Thrombocytes aid in blood coagulation 55 , but they also play an important role in the immune activity of elasmobranchs 13 .
There was no peroxidase reaction in any of the blood cells of P. wallacei, P. motoro or P. aiereba. Peroxidase is an important lysosomal enzyme involved in intracellular digestion, and one of its main features is that it indicates the absence of eosinophilic and neutrophilic granulocytes in the species investigated here 26 . However, this lack of peroxidase may be accompanied by compensatory development of other antibacterial components, such as cationic proteins 13,38 .
Though basophil leukocytes were rarely observed in the blood of P. wallacei, P. motoro and P. aiereba, their existence was confirmed through metachromasia staining. In addition, the three potamotrygonids demonstrated the presence of lipids in thrombocytes and lymphocytes but to a lesser degree than that in heterophils. Similarly, in Xiphophorus helleri (Heckel 1848), a Sudan black reaction was also demonstrated in monocytes and lymphocytes 56 . However, in other teleosts, this reaction has been observed in neutrophil granules 36 . 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 36 . The heterophils and basophils of P. wallacei, P. motoro and P. aiereba were positive for bromophenol blue staining, similar to what had previously been found in eosinophils from S. brasiliensis 36 in Amazonian turtles 26 . Positive staining was observed in basophils, eosinophils and neutrophils from P. motoro 19 . Therefore, these results indicate that these proteins play an important role in the innate defense of animals, which is possibly performed by heterophil and basophil granulocytes.
Ultrastructural analysis. The ultrastructural analyses of leukocytes from P. wallacei, P. motoro and P. aiereba were similar to each other and comparable with the findings from the sharks G. cirratum 57 and S. canicula 47 . 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 stingray leukocytes to provide basic information about these cells and make correlations with health conditions. In this manner, leukocytes can be quantified in these stingrays, which are extremely important for the aquarium industry. The cytochemical characteristics of heterophils indicates that these major granulocytes are important in the immune defense of Amazonian potamotrygonids. The blood www.nature.com/scientificreports/ cell features of wild native stingrays may be useful for making diagnoses and comparisons among these same species under controlled conditions.

Data availability
Data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. www.nature.com/scientificreports/