Influence of the Level of the Middle River Negro in the Amazon, Brazil, on the Properties of the Blood of the Cururu Freshwater Stingray Potamotrygon wallacei

Abstract

Amazonian fishes, as an adaptive form to the annual flood cycle, develop physiological strategies to adjust to variations in their habitats. The results of this study help to understand how freshwater stingrays adapt to changes in river levels and allow us to predict the physiology of blood and water properties in situations of extreme droughts and floods in rivers. This study aimed to evaluate the physiological characteristics of the freshwater stingray Potamotrygon wallacei in response to seasonal variations in the Middle River Negro, analyzing the effects of these changes on its hematological and biochemical parameters and investigating the relationship between these changes and the physicochemical composition of the water. The animals were captured in lakes and marshes in the Mariuá Archipelago in River Negro. Five field collections were carried out during periods of different flood pulses. Blood was collected by puncture of the gill vessel after the animals were anesthetized. Hematological parameters were determined by routine methods for stingrays. Blood parameters reveal close relationships with changes in river levels, which occur throughout a hydrological cycle in the Middle River Negro region. Therefore, this indicates that the hematology of P. wallacei can be used in monitoring, indicating modifications of adverse environmental changes; however, this ecophysiological association is a complex process and needs to be further investigated.

1. Introduction

The Amazon region is home to the world’s most extensive river system, characterized by a diverse mosaic of lakes interconnected by floodplains, which supports a rich biodiversity described as the largest on the planet [1]. Among the rivers that make up this system is the Negro River, which is the second largest tributary in terms of water volume, with an area of 712,000 km², and is the central blackwater system in the region [2].

The hydrological dynamics of this system are regulated by the flood pulse that controls the water cycle in the Amazon basin and establishes well-defined annual cycles, including periods of drought, flood, high water, and low water [3]. This seasonality is characterized by significant variations in water levels, altering ecological parameters, creating diverse environments, and consequently promoting the continuous adaptation of organisms [3,4,5].

Freshwater stingrays, also known as potamotrigonines, are elasmobranchs native to South America and only found in freshwater habitats. They can be found in various freshwater environments, including flooded woodland regions, lakes, beaches, and small coves with stony, muddy, or leafy bottoms [6,7]. The slow growth, long lifespan, complex reproductive patterns, and late maturation of freshwater stingrays are biological traits common to elasmobranchs. These traits lead to low population renewal rates and, as a result, increased susceptibility to the effects of commercial exploitation [8]. Therefore, the populations of these South American-only elasmobranchs are endangered by the use of freshwater stingrays for both the aquarium trade (for baby animals) and as food for adult animals. In addition, it allows the proliferation of different species, including Potamotrygon wallacei (Carvalho, Rosa, & Araújo, 2016), commonly known locally as the cururu stingray.

The stingray P. wallacei is endemic to the River Negro basin, occurring in litter environments with low water flow and currents, typical of igapó areas (submerged vegetation in the Amazon) [6,7]. P. wallacei has a reproductive cycle regulated by the river level, with copulation during the low water period and birth during the dry period [9], and has generalist feeding habits, being able to consume crustaceans, insects, and small fish [10], depending on the availability of the environment. This species is subject to substantial changes in its natural habitat due to the constant variations in the water level of the River Negro [11]. These variations significantly impact the homeostasis of organisms, causing changes in hematological and biochemical parameters [12,13].

In this context, P. wallacei may present specific physiological adaptations to maintain its homeostasis in the face of seasonal fluctuations in the River Negro. However, there is still little information on how these variations impact physiology. Thus, evaluating blood physiology responses in association with water properties and variations in water volume is extremely valuable in understanding the physiological behavior of freshwater stingrays in response to natural changes that occur in Amazonian rivers. This study’s results help us understand how freshwater stingrays adapt to river-level changes and allow us to predict the physiology of blood and water properties in extreme river droughts and floods. Therefore, the present study aims to evaluate the physiological characteristics of the freshwater stingray P. wallacei cururu in the face of seasonal variations in the level of the Middle River Negro, analyzing the effects of these changes on its hematological and biochemical parameters, in addition to investigating the possible relationships between these physiological changes and the physicochemical composition of the water.

2. Materials and Methods

2.1. Study Area

The Mariuá Archipelago is the world’s largest group of freshwater islands, comprising over 1600 islands. This archipelago is home to several species of stingrays from the Potamotrygoninae subfamily, including P. wallacei. These stingrays were captured in lakes, streams, and swamps of this archipelago in the Middle River Negro, near the municipality of Barcelos, Amazonas, Brazil. Barcelos is 436 km from the capital of the state of Amazonas (Manaus) and is known internationally as the city of ornamental fish, including freshwater stingrays.

This study was conducted according to the principles established by the Animal Ethics Committee of the Federal Institute of Education, Science, and Technology of Amazonas (1955.0905/2023 approved on 5 January 2023). All experiments were conducted according to local and ARRIVE guidelines [14], which are concerned with animal welfare and the natural maintenance of wild animal populations.

2.2. Capture and Collection of Blood from Animals

Five collections were carried out, two in December (December 1 and December 2) in different years, and one in March, June, and October, completing an entire cycle of the River Negro level, with intervals of two to three months between one collection and another. In total, 137 subadult and adult stingrays (December 1 = 25; March = 37; October = 27; and December 2 = 48) were collected during one year.

The animals were captured in lakes and flooded forests using a hand net adapted for this purpose. The stingrays were captured at night, and the fishermen traveled in canoes to the areas where the stingrays were found. The fishermen located the stingrays using headlamps, carefully moved toward them, and captured the animals by surrounding them with a hand net. After being captured, the animals were anesthetized with MS-222 (0.2 g L⁻¹), and blood was collected by puncture in the gill vessel with syringes containing needles and insertion into the center of the third branchial cleft following the recommendations of [15] with syringes containing 10% EDTA [16]. All animals were returned to the capture site after apparent recovery from anesthesia (approximately 60 min), confirmed by gill beats.

2.3. Hematological Procedures

Blood samples from captured adult and subadult stingrays were used to determine the complete blood. Initially, procedures were performed to determine the total erythrocyte count (RBC) in a hemocytometer after dilution in formalin citrate, and the count was conducted in a Neubauer chamber with the aid of a binocular optical microscope. Hematocrit (Ht) was determined using a microhematocrit capillary tube, and centrifugation was conducted, with the subsequent reading on standardized cards and the determination varying from 0% to 100%. Hemoglobin concentration (Hb) was determined by the cyanomethemoglobin method using Drabkin’s solution, and reading absorbance was conducted using a spectrophotometer. After this, the calculation of the hematimetric indices was performed: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), using the RBC, Ht, and Hb values from pre-determined mathematical formulas.

In addition, blood smears were prepared and later stained with May–Grunwald–Giemsa–Wright stain [17] before the blades had been cleaned with a suitable solution to remove grease and dirt, which was based on counting under a binocular optical microscope. These were used for the total thrombocyte count and total and differential leukocyte counts [17].

A portion of the blood was centrifuged at 750× g, and the plasma was immediately frozen in liquid nitrogen (−86 °C) and kept in this condition until the determination of the biochemical parameters. The glucose, triglycerides, total cholesterol, and urea concentrations were determined using enzymatic–colorimetric methods, quantified by commercial kits (Doles, Goiânia, Brazil) specific for each parameter. Total proteins were determined by the biuret method (Doles, GO, Brazil). The plasma levels of sodium (Na⁺) and potassium (K⁺) were analyzed by flame photometry (Micronal b 462, São Paulo, Brazil), and the levels of chloride (Cl⁻) were determined by a colorimetric method using a commercial kit (Doles, GO, Brazil). All analyses were read at light waves determined according to the specific kit and using a spectrophotometer.

2.4. Water Analysis

In each location visited where the rays were captured, the levels of dissolved oxygen and temperature (Hanna HI 9147 oximeter, São Paulo, Brazil), conductivity, and hydrogen potential (pH-meter WTW 315 i) were determined. The determination was made by inserting specific multiparameter probes, and after approximately 5 min, the quantification of the analyzed variables occurred.

Water samples were collected and stored to analyze hardness, alkalinity, ammonia, and nitrite, following previously established methods [18]. The levels of Na⁺ and K⁺ in the water were determined by flame photometry (Micronal, b 462).

2.5. Water Level of the Middle Negro River

Seasonal data on water levels of the Middle Negro River were obtained monthly from the Mineral Resources Research Company (CPRM, Manaus, Brazil), which is the body responsible for hydrological levels in Brazil.

2.6. Statistical Analysis

Analysis of variance (ANOVA), followed by Tukey’s test, considering p < 0.05, was used to test seasonal differences (dry season in March, flood season in June, beginning of low tide in October, and low tide in December) in the physical–chemical analyses, and the hematological parameters were investigated. Multiple regression was used to verify the relationships between the river water level, physical–chemical parameters of the water, and blood parameters, which were considered significant when p < 0.05.

3. Results

It was possible to collect stingrays in four of the five collections carried out. Therefore, due to the increase in water volume and the disappearance of the igapó areas, it was impossible to capture freshwater rays during the most excellent water volume period. However, the physical–chemical parameters of the water were determined in all field samples. The physical and chemical parameters of the water during the different seasonal periods are shown in

Table 1. Seasonal variation (mean ± standard deviation) of the physical–chemical parameters of the capture sites of the freshwater stingrays Potamotrygon wallacei in the waters of the Middle River Negro, Amazonas, Brazil.

Parameters	December/1	March	June	October	December/2
River level (m)	4.6 ± 0.03 a	1.6 ± 00.5 b	8.1 ± 0.09 c	3.8 ± 0.14 d	4.9 ± 7.2 e
Dissolved oxygen (mg L−1)	4.5 ± 0.3 a	4.6 ± 0.1 a	3.7 ± 0.2 b	4.0 ± 0.1 c	4.2 ± 0.3 d
Temperature (°C)	29.8 ± 0.9 a	29.8 ± 0.2 ab	26.5 ± 0.5 c	27.6 ± 0.5 d	29.4 ± 0.6 b
Conductivity (µS cm−1)	15.1 ± 1.1 e	16.9 ± 0.8 d	11.1 ± 0.7 abc	11.8 ± 0.6 b	10.9 ± 0.6 c
pH	4.2 ± 0.1 d	4.2 ± 0.1 d	4.0 ± 0.1 abc	4.0 ± 0.0 b	4.0 ± 0.1 c
Hardness (mg L−1)	2.1 ± 0.8 b	2.7 ± 0.3 b	12.6 ± 2.7 a	13.0 ± 7.6 a	13.3 ± 8.6 a
Alkalinity (mg L−1)	1.7 ± 1.1 c	4.7 ± 0.4 b	21.8 ± 2.8 a	24.4 ± 5.3 a	13.6 ± 5.6 d
Ammonia (mg L−1)	0.03 ± 0.02 d	0.04 ± 0.01 d	0.73 ± 0.08 abc	0.70 ± 0.06 b	0.75 ± 0.09 c
Nitrite (mg L−1)	0.015 ± 0.003 c	0.008 ± 0.001 a	0.009 ± 0.006 a	0.003 ± 0.001 b	0.004 ± 0.003 b
Na+ (mEq L−1)	21.0 ± 6.9 c	10.1 ± 0.7 b	71.1 ± 10.9 a	72.2 ± 16.1 a	28.3 ± 13.8 c
K+ (mEq L−1)	11.0 ± 3.3 a	9.8 ± 1.1 a	11.7 ± 1.7 a	16.3 ± 3.2 b	21.6 ± 4.1 c

Different letters in the same line indicate statistical differences (p < 0.05).

. This indicates that the lowest level of the river occurred in March, the dry season, while the highest level was observed in June, the flood season in the Middle River Negro. At the beginning of the low tide, the lowest temperatures, dissolved oxygen levels, conductivity, and the highest Na⁺ concentration were observed in June and October. However, the highest ammonia levels occurred in June and December during low tides.

Changes in glucose levels in freshwater stingrays P. wallacei occur only during the river’s dry season (March). Still, total cholesterol levels were lower on December 2 (when water levels were low) than on December 1 and in March. The values of triglycerides, total proteins, and urea did not show statistically significant differences according to the capture period of the freshwater stingrays P. wallacei (

Table 2. Seasonal variation (mean ± standard deviation) of plasma biochemical parameters of freshwater stingrays Potamotrygon wallacei collected in the Middle River Negro, in different seasonal periods.

Parameters	N	December/1	N	March	N	October	N	December/2
Glucose (mg dL−1)	25	33.2 ± 12.0 a	36	22.0 ± 9.3 b	23	34.4 ± 8.6 a	40	28.2 ± 9.9 a
Triglycerides (mg dL−1)	23	56.9 ± 18.8 a	36	57.1 ± 22.9 a	22	53.4 ± 16.8 a	34	53.2 ± 19.4 a
Cholesterol (mg dL−1)	24	61.9 ± 25.9 a	36	64.5 ± 26.4 a	22	51.8 ± 18.5 ab	36	42.1 ± 21.0 b
Total protein (g dL−1)	25	1.0 ± 0.3 a	36	1.1 ± 0.3 a	21	1.1 ± 0.3 a	34	1.0 ± 0.14 a
Urea (mg dL−1)	22	4.9 ± 5.5 a	36	5.0 ± 5.1 a	22	4.2 ± 3.4 a	32	3.0 ± 3.0 a
Chloride (mmol L−1)	24	138.8 ± 16.5 ab	30	144.7 ± 21.9 a	20	129.3 ± 22.9 ab	27	125.2 ± 18.5 b
Na+ (mmol L−1)	24	179.7 ± 44.9 a	37	191.9 ± 36.6 ab	20	216.16 ± 45.8 b	24	209.0 ± 48.7 ab
K+ (mmol/L)	24	9.1 ± 2.9 ab	37	7.4 ± 2.3 a	20	9.5 ± 3.4 b	25	9.3 ± 2.9 ab

Different letters in the same line indicate statistical differences (p < 0.05); Na⁺: sodium; K⁺: potassium.

). Plasma Na⁺ levels were lower in December/1 than in October, but K⁺ levels were lower in March than October (Table 2).

The Ht and RBC values of freshwater stingrays P. wallacei increased in the period from October to December, coinciding with the beginning of the low water level in the Middle River Negro. However, the hemoglobin concentration increased in March (during the dry season), while the MCV decreased in October. The MCHC increased in March, October, and December, but the MHC increased in March and October (Figure 1). Thus, the increase in river volume appears to be a decisive factor in promoting changes in Ht, Hb, RBC, MCHC, MVH, and MCH values.

The number of thrombocytes underwent no seasonal change (

Table 3. Seasonal variation (mean ± standard deviation) of the thrombogram and leukogram of freshwater stingray Potamotrygon wallacei collected in the Middle River Negro in different seasonal periods.

Parameters (µL)	December/1 N = 25	March N = 36	October N = 26	December/2 N = 40
Thrombocytes	1181 ± 602 a	1492 ± 1629 a	987 ± 1068 a	1032 ± 986 a
Leukocytes	5615 ± 3668 ab	3367 ± 2110 a	6426 ± 2657 b	4499 ± 3443 ab
Lymphocytes	2324 ± 1379 a	1627 ± 1355 a	2321 ± 1553 a	1937 ± 1733 a
Monocytes	1760 ± 1148 ac	882 ± 626 a	2308 ± 1184 b	1231 ± 1008 bc
Heterophils	735 ± 345 a	738 ± 463 a	1171 ± 539 a	1082 ± 731 a
Basophils	202 ± 153 a	95 ± 135 a	128 ± 165 a	138 ± 141 a

Different letters in the same row indicate statistically significant differences (p < 0.05).

). This indicates that this cell type in the blood of the freshwater stingray P. wallacei is not subject to changes caused by the river level or changes in water properties. However, the total leukocyte and monocyte counts were higher in October than in March (Table 3). These leukocytes in the cururu stingray are sentinels for changes caused by the flood pulse. No statistically significant differences were observed in the different months of freshwater stingray collections for the values of lymphocytes, heterophils, and basophils.

Table 4. p-values in the multiple regression for the factors influencing the hematological parameters of freshwater stingray Potamotrygon wallacei collected in the Middle River Negro, Amazonas, Brazil.

Parameters	Glucose	Cholesterol	Chloride	Na+	K+	Leukocytes	Monocytes	Ht	Hb	RBC	MVC	MHC	MCHC
River level	0.099	0.012 *	0.004 *	0.918	0.708	0.283	0.364	0.016 *	0.003 *	0.001 *	0.669	0.188	0.049 *
Dissolved oxygen	0.372	0.520	0.535	0.899	0.249	0.823	0.333	0.091	0.771	0.531	0.383	0.903	0.183
Conductivity	0.198	0.216	0.027 *	0.546	0.026 *	0.001 *	0.001 *	0.004 *	0.087	0.003 *	0.683	0.930	0.798
pH	0.428	0.303	0.883	0.637	0.548	0.062	0.727	0.834	0.193	0.325	0.502	0.359	0.063
Temperature	0.097	0.843	0.170	0.606	0.838	0.993	0.259	0.477	0.296	0.047 *	0.761	0.096	0.186
Hardness	0.503	0.110	0.336	0.101	0.895	0.044 *	0.067	0.370	0.468	0.087	0.902	0.742	0.870
Alkalinity	0.001 *	0.002 *	0.060	0.954	0.021 *	0.002 *	0.005 *	0.000 *	0.326	0.078	0.471	0.661	0.412
Ammonia	0.301	0.101	0.663	0.970	0.107	0.005 *	0.012 *	0.586	0.294	0.017 *	0.152	0.343	0.785
Nitrite	0.000 *	0.022 *	0.001 *	0.202	0.130	0.021 *	0.051	0.003 *	0.660	0.001 *	0.523	0.100	0.239
Na+	0.064	0.136	0.001 *	0.435	0.009 *	0.346	0.324	0.001 *	0.166	0.021 *	0.001 *	0.024 *	0.846
K+	0.080	0.050	0.098	0.658	0.326	0.792	0.994	0.174	0.587	0.750	0.346	0.503	0.284

* indicates significant influence (p < 0.05). Na⁺: sodium; K⁺: potassium; Ht: hematocrit; Hb: hemoglobin concentration; RBC: red blood cell count; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration.

presents the results of multiple regressions between river levels and water’s physical and chemical properties in relation to blood physiological parameters. The relationships are significant when the p-value is less than 0.05.

Of the variables related to water, a relationship was observed between river level (six variables), conductivity (six variables), temperature (one variable), hardness (one variable), alkalinity (six variables), ammonia (three variables), nitrite (six variables) and Na⁺ (six variables) (Table 4). No relationship was observed in the water parameters of dissolved oxygen, pH, and K⁺ (Table 4).

In the blood parameters of the freshwater stingray P. wallacei, a relationship was observed in the values of glucose (two variables), cholesterol (three variables), chloride ( four variables), K⁺ (three variables), leukocytes (five variables), monocytes (three variables), Ht (five variables), Hb (one variable), RBC (six variables), MVC (one variable), MHC (one variable), and MCHC (one variable).

4. Discussion

Studies on the hematology of freshwater stingrays reveal that blood parameter values can be influenced by variations in water parameters, such as water temperature, oxygen concentration, pH, and ion concentration and types, in addition to influencing preference for habitat occupation and use [5,6,7,12,13,19,20]. Another factor that can cause hematological changes is using anesthetics, which can be chemical stressors in fish [21]. In the present study, the anesthetic used was MS-222, which can cause lesions in the gills and consequently interfere with values such as Ht, RBC, and Hb since the blood loss caused by the lesions can interfere with the erythrogram [22]. Changes in glucose, cortisol, and lactate levels may also occur, which are stress indicators [23]. In the present study, possible effects were suppressed due to the standardization of the use of blood collections in all research phases.

For this reason, the water quality of the Middle River Negro was determined in this study, and temperature variations, dissolved oxygen levels, electrical conductivity, ammonia, and nitrite, as well as in the water level of the Middle River Negro were observed. Inadequate water quality conditions impair organisms’ growth, reproduction, health, and survival [24].

During the sampling period, when the river was at its highest elevation, it was not possible to capture individuals of P. wallacei due to the increase in water volume and the preferred environment of this species being flooded by the river pulse, in addition to the reduction in transparency, which causes low visibility and makes capture difficult [25,26]. In addition, factors related to water quality, such as low levels of dissolved oxygen and temperature, were reported during this period, conditions that make it difficult for this species to become accustomed to and even for teleost fish to be present in shallower waters.

Potamotrigonines have adaptation strategies to live in black water poor in ions that are similar to those of teleosts in the River Negro, that is, it involves a low-affinity ion transport system [27]. Na⁺/K⁺ ATPase activities in the gills and kidneys are higher in fish that inhabit waters with low ion concentrations, facilitating the efficient uptake and retention of these elements [27]. Although Na⁺ and K⁺ levels varied in some periods of the river cycle, the values observed, in general, were similar to other studies on stingray physiology, including Potamotrygon motoro, Paratrygon aiereba, and P. wallacei in the River Negro basin [6], as well as P. motoro and Potamotrygon orbignyi in the Solimões River, the river of white water and Potamotrygon falkineri and P. motoro in the Paraná River basin and Potamotrygon scobina and P. orbignyi in the Piririm River [11,12]. This suggests osmoregulatory mechanisms in freshwater rays exhibit a conserved pattern across species, and this is a fundamental adaptive characteristic for living in environments that experience seasonal variability. These adaptations allow the preservation of ionic balance in adverse environments; some species adjust their physiological osmoregulation mechanisms according to the chemical composition and type of water [26].

Duncan and Fernandez [27], when evaluating the distribution pattern of freshwater stingrays of the Potamotrygonidae family, observed that the different types of water in the Amazon region and the possible differences within the same river cause changes in the physical and chemical characteristics of the waters of the Amazon basin. Thus, they can act as barriers or hydrological filters for the dispersion of freshwater species from the Amazon region [27].

The levels of glucose, cholesterol, triglycerides, proteins, urea, and chloride were similar to those reported for several species of freshwater stingrays in the Amazon basin [7,8,9,10,11,19,20,21,22,23,24,25,26]. However, in the present study of P. wallacei, plasma glucose concentrations decreased during the dry season, and statistical analyses demonstrate the influence of alkalinity and nitrite on glycemia. However, during this period, there is less food availability in a more restricted environment for this slow species, which competes with other, more active species, such as some predatory teleosts. Thus, it spends more time searching for food. Still, this temporary food deprivation cannot alter secondary metabolic pathways, such as total cholesterol and triglycerides, or affect total protein levels. There were also fluctuations in cholesterol levels in P. wallacei statistically related to river water levels, alkalinity, and nitrite concentration. However, this correlation must be causal since variations in plasma lipids are more intrinsically linked to sexual maturation, reproductive interval, nutritional behavior, metabolic adaptation, and intense growth than environmental conditions [28,29].

The Ht and RBC of P. wallacei showed a significant increase between October and December. The MVC decreased in October, and the MCHC increased in October and November. This increase indicates an increase in erythropoiesis during these periods since the number of erythrocytes was correlated with temperature and other environmental conditions such as river level, conductivity, ammonia, and nitrite. With the increase in erythropoiesis, the rays can compensate for the increased demand for dissolved oxygen since oxygen levels decrease during the low tide/dry period [12,29]. Fish blood typically contains heterogeneous populations of erythrocytes with immature cells, initially small and with less hemoglobin than more mature cells [29]. Therefore, the increase in Ht and RBC in cururu ray P. wallacei corresponds to an intensification of erythropoiesis and the production of new erythrocytes with a smaller volume but a lower hemoglobin concentration.

In P. wallacei, at the beginning of low tide in October, there was an increase in the total number of leukocytes, primarily due to the rise in monocytes. Statistical analysis reveals that electrical conductivity, hardness, alkalinity, ammonia, and nitrite levels influenced the number of leukocytes. Therefore, the seasonal variation in the number of total leukocytes for the cururu ray P. wallacei appears to be related to its ecophysiological conditions. Research indicates that the number of leukocytes also responds to changes in water quality [5,6]. However, differences in the number of leukocytes can also be attributed to biotic factors, such as age, sex, stress, nutritional status, and pathogens [5,6,7]. Furthermore, observed an increase in the number of intraerythrocytic parasites during the dry season in populations of P. wallacei in the Middle River Negro region, indicating that the river level influences the incidence of hemoparasites and the dry season is the period of most significant susceptibility to infestation [11].

In the multiple regressions, associations were observed mainly in the river level, conductivity, alkalinity, nitrite, and Na⁺, indicating that these water variables actively influence the characteristics of the blood physiology of the freshwater stingray P. wallacei. Furthermore, more pronounced relationships were observed in leukocytes, Ht, and the RBC for the blood parameters, demonstrating that the flood pulse most influences the red blood series in rivers in the Amazon region.

The results of the present study provide fundamental and relevant information about cururu ray P. wallacei, a significant Amazonian species, primarily due to its considerable interest as an ornamental fish. As expected, the erythrocyte parameters exhibited a close relationship with changes in river levels and, consequently, in environmental conditions, a phenomenon also observed during the reproductive period, which is strongly regulated according to water levels [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23].

5. Conclusions

As with other species of Amazonian teleost fish, the blood parameters of the cururu P. wallacei freshwater stingray reveal a close relationship with changes in river levels, which occur throughout a hydrological cycle in the Middle River Negro region, Amazonas, Brazil. Therefore, this indicates that the hematology of the cururu stingray P. wallacei can be used to monitor changes in response to adverse environmental conditions. However, this ecophysiological association is a complex process that needs further investigation and must be evaluated regularly, mainly because extreme drought events have occurred in the Amazon region’s rivers in recent years.

Furthermore, changes in the blood physiology of the cururu stingray P. wallacei demonstrate a substantial relationship with the changes caused by the flood pulse of the Amazon rivers. Thus, the alarming recurring droughts of the rivers in the Amazon may threaten the survival of the stingray P. wallacei, which is classified as an endemic species of the Middle River Negro.