Ray Transport During the Sampling Individuals of P

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy

Comparative Biochemistry and Physiology, Part A 162 (2012) 139–145

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A

journal homepage: www.elsevier.com/locate/cbpa

Stress responses of the endemic freshwater cururu stingray (Potamotrygon cf. histrix) during transportation in the Amazon region of the Rio Negro☆

R.P. Brinn a,⁎, J.L. Marcon b, D.M. McComb c, L.C. Gomes d, J.S. Abreu e, B. Baldisseroto f a Florida International University, 3000 NE 151 st. 33181, Miami, FL, USA b Universidade Federal do Amazonas (UFAM), Av. General Rodrigo Octávio Jordão Ramos, 3000, Campus Universitário, Coroado I, 69077-000, Manaus, AM, Brazil c Harbor Branch Oceanographic Institute at Florida Atlantic University, 34946, Fort Pierce, FL, USA d Centro Universitário Vila Velha, Vila Velha, ES, Brazil, Rua Comissário José Dantas de Melo, 21, Boa Vista, ,29101-770 Vila Velha, ES, Brazil e Universidade Federal de Mato Grosso, Faculdade de Agronomia e Medicina Veterinária (FAMEV), Avenida Fernando Corrêa da Costa, 2367, Boa Esperança, 78060-900, Cuiaba, MT, Brazil f Universidade Federal de Santa Maria, Campus Camobi, 97105-900, Santa Maria, RS, Brazil article info abstract

Article history: Potamotrygon cf. histrix (cururu stingray) are endemic freshwater stingrays from the middle region of the Rio Received 28 February 2011 Negro in the Brazilian Amazon basin and are exported worldwide as ornamentals caught by artisanal Received in revised form 2 July 2011 fishermen. The transport process from capture to final destination is long and stressful. This study quantified Accepted 5 July 2011 stress related changes in corticosterone, blood and water samples (baseline, pre-transport, 3 h, 12 h and 24 h) Available online 13 July 2011 analyzed during a transport experiment which tested two water additives (tetracycline and the probiotic fi fi Keywords: E nol®). There was a signi cant stepwise increase in corticosterone levels in stingrays over transport time in Amazonian stingrays combination with osmoregulatory disturbances suggesting a stress related role of this corticosteroid. There + + Corticosterone were significant increases in water conductivity, Na and K losses and ammonia excretion. Blood Elasmobranch parameters such as glucose, hematocrit, red blood count and urea did not change significantly during the Osmoregulation experiment. Glucose levels did not increase significantly during transport and this may be due to the fact that Transport stress other elasmobranchs have been shown to rely more on ketone bodies for energy rather than glucose and produce ammonia as their main nitrogenous waste. The mineralocorticoid action of this hormone has been shown in elasmobranchs and most likely plays a role in osmotic homeostasis. The use of probiotic and especially antibiotic should be avoided since no beneficial effects were observed. © 2011 Elsevier Inc. All rights reserved.

1. Introduction The Brazilian Amazon region is home to a pigmy freshwater stingray that is endemic to a small portion of the Rio Negro, a large There are over 800 extant elasmobranch species most of which affluent of the Amazon River. Five stingray species are legally exported inhabit marine environments (Heinicke et al., 2009). Of the 400 batoid from the Brazilian Amazon basin under government managed annual (skates and rays) species at least 20 Potamotrygonid rays are quotas, including Potamotrygon cf histrix, regionally called “Cururu exclusively freshwater and distributed among four genera: Heliotrygon Ray”, which has yet to be fully described. Freshwater stingrays are (two species), Paratrygon (one species), Plesiotrygon (two species) and targeted by the ornamental industry primarily due to high economic Potamotrygon (18 valid species) (Carvalho et al., 2003; Martin, 2005; value making them vulnerable to fishing pressure. The city of Barcelos, Carvalho and Lovejoy, 2011; Carvalho and Ragno, 2011). In contrast to within the Brazilian Amazon region, is an artisanal fishing community marine species, freshwater stingrays possess unique morphological which collects and exports thousands of ornamental fishes from the and physiological specializations including low plasma urea levels, a Amazon including P.cf histrix (Chao et al., 2001). The Rio Negro is a reduction in rectal gland size (Thorson et al., 1983) and a reduction in warm, ion poor, oxygen depleted, acidic river reaching 3.5 pH in some the tubule length of the Ampullae of Lorenzini (Raschi and Mackanos, areas (Matsuo and Val, 2003; Mortatti and Probst, 2003). Small 1989; McGowan and Kajiura, 2009). streams (igarapés) feed the main Rio Negro system with tannin rich waters supporting high ichthyological diversity. The Rio Negro Basin has a high seasonal variation in water level and the stingrays have a ☆ This paper stems from a presentation in the Symposium "The Physiological Stress predetermined reproductive cycle strongly linked to the river pulse Response in Elasmobranch Fishes", at the 26th annual meeting of the American (Charvet-Almeida et al., 2005). Local fishermen catch, collect and Elasmobranch Society, held on July 11, 2010, in Providence, Rhode Island (USA). transport stingrays with rudimentary techniques and in many cases ⁎ Corresponding author. Tel.: +1 305 919 5348; fax: +1 305 919 4040. add unmeasured and untested compounds including tetracycline to E-mail addresses: brinnr@fiu.edu (R.P. Brinn), [email protected] (J.L. Marcon), [email protected] (D.M. McComb), [email protected] (L.C. Gomes), the water in an effort to minimize stress and mortality. Once taken [email protected] (J.S. Abreu), [email protected] (B. Baldisseroto). from the wild and transported to Barcelos, the rays are carried by

140 R.P. Brinn et al. / Comparative Biochemistry and Physiology, Part A 162 (2012) 139–145 freight boat to Manaus in transport that can last over 24 h dependent and 24 h of transport. Each box was sampled only once and then upon river conditions. The transport occurs in overcrowded plastic discarded from the experiment thereafter. totes with little water to optimize space and cost, creating unfavorable conditions to fish. Techniques developed to minimize stress during transport in teleosts have been successful in other species and include 2.3. Blood collection and analyses the addition of probiotic products (Gomes et al., 2008; 2009). Once arriving in Manaus, fish are held in quarantine at local exporters prior After anesthesia and tail immobilization stingrays were kept in to international exportation. Many stingrays are subsequently flown to ventral position and a cardiac blood sample taken with an insulin-type Miami (FL, USA) where they are held for weeks or months prior to final hypodermic 1 mL syringe coated with ETDA 10% as anticoagulant. To shipment worldwide. The transport process is lengthy and therefore ensure no mortality of experimental animals due to excessive blood acute and chronic stress strongly impacts survivorship. loss, the volume removed was less than 1% of the individual body mass. Measuring stress in elasmobranchs has been a challenge because The same procedures were applied for all samples collected in all they produce a unique stress hormone identified as 1 α hydroxicorti- treatment groups. Blood was used for hematocrit (Htc) determination costerone (Idler and Truscott, 1966) which has no commercially by the microhematocrit centrifugation technique, and for red blood available measuring technique. Some species of elasmobranch have cell counts (RBC) performed by manual counting in a Neubauer also been shown to produce corticosterone however with no well- hemocytometer with 1/200 dilution of blood in phormol-citrate defined stress related role (Truscott and Idler, 1972; Rasmussen and solution. MCV (mean cellular volume) was calculated based on the Gruber, 1990; Manire et al., 2007). obtained Htc and RBC values. The remaining blood was centrifuged at The primary objective of this experiment was to investigate stress 2500 g for 3 min and plasma was immediately stored in a liquid related osmoregulatory, blood parameter alterations and costicoster- nitrogen container (Cryo Diffusion S.A., France). one responses of P. cf. histrix (cururu stingray) to two different water Plasma samples were analyzed for glucose, urea, chloride levels additives during the transport process. The secondary objective was and osmolarity. Glucose levels were measured by the enzymatic to contribute to the general understanding of the stress response in method of glucose oxidase at 510 nm and Urea was determined by freshwater elasmobranchs. an enzymatic method (600 nm) based on the reaction with urease in alkaline medium. The concentration of chloride ions was quantified by a coulometric titration method using a digital chloridometer 2. Materials and methods (Labconco Corp., USA), and plasma osmolarity was measured using a mercury vapor pressure osmometer (Wescor Inc., USA). 2.1. Study area and animal capture

Specimens of cururu stingray, P. cf. histrix (n=69, weight=240.29± 2.4. Corticosterone assay 137.29 g; disc width=17.14±2.80 cm; mean±SD; 26 females and 43 males) were captured by professional fishermen in areas of flooded Serum corticosterone levels were determined through radioim- forest covering the margins of Igarapé Puxurituba near Barcelos, munoassay method using a coat-a-count kit (DPC, USA) developed for (Amazonas, Brazil) with hand nets typically at night as stingrays were rat corticosterone. Levels were validated using parallelism and intra detected using flashlight eye shine. Once captured they were immedi- and interassays values that were found not to exceed a 15% variation. ately placed in holding boxes and then later transferred to 1 m3 net Lemon shark (Negaprion brevirostris) plasma samples from sharks pens, where fish were maintained for a maximum of 10 days with collected in Marquesas, FL USA (2003) in an unrelated trip were also continuous natural water flow, and fed daily fresh shrimp and fish analyzed to compare the concentration of corticosterone between a collected in the surrounding areas. Feeding ceased 24 h prior to the marine species with described levels (Rasmussen and Gruber, 1993) beginning of the transport experiment. Field experiments and transport and the unknown levels of this freshwater elasmobranch. occurred in November of 2005 corresponding to the dry season in the middle Rio Negro basin. 2.5. Water quality and net ion fluxes

2.2. Experimental transport protocol The water parameters, temperature, dissolved oxygen levels (YSI 55 meter, USA), pH (YSI pH-100 meter, USA) and electric conductivity Since there are no baseline serum profiles established for free (Bernauer digital meter, Brazil), were measured directly from the ranging Amazonian freshwater stingrays, a baseline group (B) of nine plastic boxes used for stingray transport during the sampling individuals of P. cf. histrix were collected, anesthetized with benzo- procedure. Additional water samples were taken for further analysis caine (100 mg L−1) and blood drawn under 3 min to avoid possible of alkalinity by titration (American Public Health Association, 1992), + handling effects in corticosterone levels. To test for potential stress total ammonia (NH3 +NH4 ) determined by the salicylate method effects of net pen confinement another group of stingrays (n=6) held (Verdouw et al., 1978), and net ion fluxes of Na+,K+ and Cl−. in netpens were sampled just before transport experiment (BT). For Water Na+ and K+ levels were measured directly in a B462 flame the transport experiment, the remaining individuals were randomly photometer (Micronal, Brazil) and Cl− levels by the colorimetric assay distributed in 54 plastic boxes with a total capacity of 40 L, but filled described by Zall et al. (1956). Net ion fluxes (Jnet) were calculated with 10 L of local stream water, and transported in three different based on changes in the ion concentration of transported water over treatment conditions (n=6 for each combination of time and the sampling periods according to the equation by Gonzalez et al. treatment): a control group with no additive (NO-ADD), and two (1998): Jnet=V([ion]1−[ion]2).(Mt)−1, where [ion]1 and [ion]2 other groups maintained in a diluted solution of probiotic (20 mg L−1; are the bath ion concentrations at the beginning and end of the flux PB; Efinol®L, Bentoli Agrinutrition Inc., TX, USA) with a concentration period, respectively, V is the bath volume in liters, M is the mass of the in accordance with the manufacturer, and antibiotic tetracycline fish in kg, and t is the duration of the flux period in h. (200 mg L−1; AB) which was estimated to be what local fishermen Waterborne ion levels (μmol L−1) in the igarapé were 7.22 Na+ use. During the 24 h of transport the vessel proceeded in conditions and 2.19 K+. Initial waterborne ion levels in the fish boxes were typical of the local trade to take fish from Barcelos to Manaus. Blood (μmol L−1, mean±SEM): Na+ 18.41±2.01, 19.12±1.87 and 31.98± parameters of stingrays, water quality and net ion fluxes were moni- 0.80; K+ 6.42 ±0.84, 12.78 ±1.44 and 11.80 ±0.45 for control, tored from six different boxes (replicates) of each treatment at 3, 12 antibiotic and probiotic groups, respectively. Author's personal copy

R.P. Brinn et al. / Comparative Biochemistry and Physiology, Part A 162 (2012) 139–145 141

2.6. Statistical analysis A 180 The data were expressed as the mean±SEM. The homogeneity of 160 * variances between groups was tested with the Levene test. All the * * ) 140 measured parameters presented homogenous variances and the data -1 were compared by two-way ANOVA (treatment x time of transport) 120 followed by the Tukey test, except plasma urea and dissolved oxygen 100 (mmol L values, which were compared by Kruskall–Wallis and multiple - 80 comparisons of mean ranks. Corticosterone and ammonia results were log transformed prior to statistical analyses. Software Statistica version 60 ﬁ 7.0 was used for statistical analysis and the minimum signi cance level 40 was set at Pb0.05. Plasma Cl 20

3. Results 0 B BT 3h 12h 24h Plasma corticosterone levels did not show any significant differ- B 500 ences due to treatment but did show significant changes over time. Corticosterone basal and pre-transport levels were significantly lower o + + from the NO-ADD, AB and PB treatment levels in all three times. 400 * * * * * * ** Stingrays exposed to antibiotic for 12 h presented significantly lower * * corticosterone levels than those exposed to antibiotic 24 h (Fig. 1). 300 Stingrays transported with antibiotic for 3 and 12 h and probiotic for 12 h presented significantly lower plasma Cl− levels than basal values (Fig. 2A). Plasma osmolarity of all treatments were significantly 200 lower than basal values, and 12 h after the beginning of the transport, plasma osmolarity was significantly higher in those transported with 100 antibiotic and probiotic than before the transport. In addition, stingrays from the control group also significantly increased plasma osmolarity Plasma osmolarity (mOsmkg) after 12 h of transport compared to the same group 3 h after transport 0 + + B BT 3h 12h 24h (Fig. 2B). Rays submitted to transportation presented Na and K net Sampling time effluxes (except those transported with antibiotic for 3 h), while those transported with probiotic for 3 h showed significantly higher net Na+ Control Antibiotic effluxes than those from the other treatments. The use of antibiotics for Probiotic 3hledtoanetK+ influx, while the other treatments showed net K+ effluxes at the same time of sampling. These differences were not Fig. 2. Plasma Cl− (A) and osmolarity (B) of the freshwater cururu stingray during observed after 12 h of transport, and in rays transported with probiotics transport procedures N=6 (except for basal, N=9). B: basal; BT: before transport; 3 h, fi net Na+ and K+ effluxes decreased after 12 and 24 h, respectively (Fig. 3). 12 h and 24 h: time after beginning of the transport. *=signi cantly different from basal values. +=significantly different from BT. 0=significantly different from the Waterborne ammonia and conductivity levels increased progressively same group at 3 h. but only after 24 h of transport and were significantly higher than basal and before transport values (Fig. 4). Fish exposed to probiotic for 24 h also presented significantly higher waterborne ammonia levels com- antibiotic exposed fish after the same time of transport. Fish exposed to pared to those exposed to probiotic for 3 h. Fish exposed to probiotic antibiotic for 3 h presented significantly higher ammonia excretion than for 3 h showed significantly lower ammonia excretion than control and those exposed for 12 and 24 h (Fig. 5). After 12 and 24 h of transport water pH was significantly higher in all treatments groups (but probiotic- exposed only 24 h) than basal and before transport values. No significant 200 + + ) + * differences were observed in glucose, Ht, RBC, MCV, urea, alkalinity, -1 * o temperature and oxygen levels (Tables 1 and 2). + * 150 4. Discussion * + This study examined stress related changes in the hormonal and 100 + * + + osmoregulatory characteristics of P. cf. histrix subjected to a transport * + fi * experiment using water additives. For the rst time corticosterone * * levels have been determined for this species and with additional 50 blood and water parameters define the physiological stress response. Baseline data will be instrumental in developing best management Plasma corticosterone (ng mL practices for the ornamental industry and for future conservation 0 B BT 3h 12h 24h studies of this endemic Amazonian stingray. Corticosterone (CS) is a highly conserved vertebrate hormone that Control Antibiotic cannot be stored intracellularly, is synthesized and circulated on Probiotic demand and is therefore a great target for stress studies. Idler and Truscott (1966) discovered a compound more polar than cortisol Fig. 1. Plasma corticosterone of the freshwater cururu stingray during transport described as 1alpha hydroxycorticosterone (1 α HCS) using interrenal procedures. N=6 (except for basal, N=9). B: basal; BT: before transport; 3 h, 12 h and incubates of the elasmobranchs Raja radiata and Raja ocellata. Several 24 h: time after beginning of the transport. *=significantly different from basal values. +=significantly different from BT. 0=significantly different from the same group at other marine elasmobranchs (Truscott and Idler, 1972; Hazon and 12 h. Henderson, 1984; Armour et al., 1993) have been shown to produce Author's personal copy

142 R.P. Brinn et al. / Comparative Biochemistry and Physiology, Part A 162 (2012) 139–145 A 0 A o+ ) 250 + -1 ) * -1 * +

h -50 -1 a ao mol L 200 * ao µ ao -100

mol kg a ao 150 µ a

-150 100 fluxes ( +

-200 a 50 Net Na Waterborne ammonia ( b -250 0 3h 12h 24h B BT 3h 12h 24h B B 100 b + 50 ) * -1 ) h 80 -1

-1 + 0 * + S cm 60 µ

mol kg * µ a a a -50 ao 40 a a fluxes ( + a Conductivity ( 20 -100 Net K a 0 3h 12h 24h BBT 3h 12h 24h Sampling time Control Control Antibiotic Antibiotic Probiotic Probiotic

Fig. 4. Total ammonia (A) and electric conductivity (B) levels of the water where cururu Fig. 3. Na+ (A) and K+ (B) net fluxes of the freshwater cururu stingray during transport stingrays were collected, maintained or during transport procedures. N=6. B: basal; procedures. N=6. 3 h, 12 h and 24 h: time after beginning of the transport. Different BT: before transport; 3 h, 12 h and 24 h: time after beginning of the transport. letters in the bars indicate significant difference between treatments in the same *=significantly different from basal values. +=significantly different from BT. sampling time. o=significantly different from the same group at 3 h.

concentrations while GR require higher concentrations (Carroll et al., this unique steroid hormone. This hormone has been a challenge to 2008). measure since no commercial kit is available and methods to isolate Our ﬁndings suggest some possible CS responsiveness to transport and synthesize (Manire et al., 2007) this hormone have not been stress in P. cf. histrix with baseline levels of 7 ng mL−1 in rays caught in achieved, therefore this hormone was not measured in Cururu. It has the wild, levels rose signiﬁcantly in pre-transport stingrays to about been suggested that corticosterone is a precursor of 1 α HCS (Idler and Truscott, 1967) and has been detected in several elasmobranchs (Idler and Truscott, 1968; Truscott and Idler, 1968; Truscott and Idler, 1972; 0 )

Kime, 1977; Rasmussen and Gruber, 1990, 1993; Rasmussen and -1 h

Crow, 1993; Snelson et al., 1997) although in smaller quantities and -1 -100 ﬂ with a con icting physiological role. b

Questions about the non-pituitary regulation of interrenal activity mol kg have been proposed (Hazon and Henderson, 1984) and dogﬁsh µ -200 ao ao (Scyliorhinus canicula) that were hypophysectomized and kept alive ao α ao for 14 months still produced 1 HCS. On the other hand while using -300 a ﬁ heterologous ACTH (porcine) the dog sh showed a dose dependent a increase in 1 α HCS (O'Toole et al., 1990). Similar results were found a for R. ocellata (Idler and Truscott, 1967) while incubating interrenal -400 tissue and adding ACTH, suggesting regulatory role of the pituitary a Ammonia excretion ( and that 1 α HCS was the major secretory product of the gland. -500 In vertebrates there are two common corticosteroid receptors 3h 12h 24h

(CR); one regulates energy metabolism, immunity and stress re- Control sponses which are glucocorticoid receptors (GR); and the others are Antibiotic Probiotic mineralocorticoid receptors (MR) which help regulate electrolyte homeostasis. Both are thought to have evolved from a single ancestral Fig. 5. Ammonia excretion of the freshwater cururu stingray during transport CR at least 470 million years ago (Thornton, 2001; Bridgham et al., procedures. N=6. 3 h, 12 h and 24 h: time after beginning of the transport. Different fi 2006). Corticosteroids have been shown to bind with low speci city to letters in the bars indicate significant difference between treatments in the same MR and GR in skates (Leucoraja erinacea) but MR is activated with low sampling time. o=significantly different from the same group at 3 h. Author's personal copy

R.P. Brinn et al. / Comparative Biochemistry and Physiology, Part A 162 (2012) 139–145 143

Table 1 previous study, probably due to the length of time that stingrays were Physical and chemical parameters of water during transport procedures measured at kept in captivity. Lower plasma Cl− and osmolarity but similar urea and sampling time. hematocrit levels in the same species were presented by Bittner and Treatments pH Temperature D.O. Alkalinity Lang (1980), although no details regarding the rays maintenance or −1 (°C) (mg L ) (ppm) collection were provided in that study. Maintenance of the rays in − Basal 4.07±0.02 27.10±0.10 2.94±0.30 10.27±0.97 netpens did not change CS and plasma Cl levels compared to basal Before transport 5.30±0.03 27.00±0.08 4.77±0.23 9.24±0.81 values, but osmolarity decreased significantly, indicating that at least Control 3 h 6.02±0.08 26.73±0.15 2.24±0.49 13.31±2.26 some osmoregulatory imbalance occurred. The highest Na+ and K+ Antibiotic 3 h 6.03±0.07 26.70±0.13 2.13±0.48 11.73±0.93 + Probiotic 3 h 5.74±0.07 26.55±0.21 3.00±0.64 10.05±0.57 losses were observed after 3 h of transportation (except K in rays Control 12 h 6.72±0.07a 26.48±0.45 4.52±0.66 9.53±1.09 exposed to antibiotics), which is in agreement with the CS increase Antibiotic 12 h 6.78±0.08a 26.37±0.17 3.09±0.64 10.27±1.23 compared to basal and before transport levels. However, in spite of CS Probiotic 12 h 6.50±0.06 26.05±0.19 2.77±0.49 10.41±0.96 levels remaining significantly higher than before transport values, Na+ Control 24 h 6.75±0.08a 28.33±0.27 2.76±0.56 15.18±2.58 and K+ losses decreased after 12 h of transportation. Plasma osmolarity Antibiotic 24 h 6.73±0.05a 28.43±0.23 1.59±0.36 16.13±1.47 Probiotic 24 h 6.65±0.09a 28.50±0.23 1.88±0.41 17.97±2.97 also did not return to basal values after 12 or 24 h of transportation. Cardinal tetras, Paracheirodon axelrodi, and marbled hatchetfish, a Significantly different from basal and before transport levels. Carnegiella strigata also collected from the middle Rio Negro and transported using similar methodology, presented similar results, i.e., a 10 ng mL−1, reaching an average of 64 ng mL−1 3 h after the transport decrease in ion losses after 12–24 h of transport (compared to 3 h) and and finally reaching 136 ng mL−1 after 24 h of transport. These levels maintenance of high cortisol levels through 24 h of transportation are 6 fold higher than any value found in other elasmobranchs (Idler (Gomes et al., 2008, 2009). The detected Na+ and K+ losses and and Truscott, 1967; Truscott and Idler, 1972; Rasmussen and Gruber, ammonia excretion rate from the stingrays can explain the significant 1990, 1993; Snelson et al., 1997; Manire et al., 2007) suggesting it may increase of water conductivity after 24 h of transport. function as a glucocorticoid hormone. Glucose increase is a secondary Certain blood variables like hematocrit and hemoglobin are response and widely used as a measurement of stress (Mazeaud and considered auxiliary stress response indicators (Morgan and Iwama, Mazeaud, 1981; Morgan and Iwama, 1997; Wendelaar Bonga, 1997)but 1997; Tavares-Dias and Moraes, 2004). In other fish species the P. cf. histrix levels did not change significantly and that is most likely due stimulatory effect of catecholamines and cortisol promote the increase to the fact that some elasmobranch species have shown an enhanced in oxygen demand in the tissues (Morgan and Iwama, 1996), reliance in the use of ketone bodies (Ballantyne, 1997) which has been demanding a quick differentiation and proliferation of erythrocytes. demonstrated in P. motoro (Speer- Roesch et al., 2006). If this is the case The lack of significant changes in the blood variables, including red cell corticosterone might be acting more like a “ketonecorticoid” since count, hematocrit and MCV, as well as in glucose levels during the studies in rats have found corresponding increases in ketone bodies course of transport might indicate that freshwater stingrays as well as with elevations of stress related corticosterone (Ricart-Jane´ et al., 2002; other elasmobranch species do not respond in the same way to the Teague et al., 2007). It is also possible that the moderate hypoxia the general stress syndrome observed in several freshwater teleosts. In the rays experienced during transport did not trigger a hormonal response present study the hematocrit values found in the basal group of or activation of glucogenolisys. In its natural environment, the flooded stingrays were similar to those found in Potamotrygon hystrix under forests and igarapés, the cururu stingray is usually exposed to low freshwater conditions (21.6%, Bittner and Lang, 1980) and in the oxygen levels. Therefore the non responsiveness of blood parameters to Atlantic stingray Dasyatis sabina (21.5%, Piermarini and Evans, 1998) transport stress must be interpreted with caution. as well as sharks species, Sphyrna tiburo (Carlson and Parsons, 2003), Secondary stress responses observed in fish include osmoregula- and Heterodontus portusjacksoni, but slightly higher than in Mustelus tory disturbances (Eddy, 1981; McDonald and Milligan, 1997; antarcticus (Frick et al., 2010). In a similar way, RBC counts and MCV Wendelaar Bonga, 1997; Benfey and Biron, 2000; Wojtaszek et al., were in the range of other elasmobranchs (Wilhelm Filho et al., 1992; 2002). Hazon and Henderson (1984) suggested that 1 α HCS has some Arnold, 2005; Brill et al., 2008). influence in the osmoregulatory functions of urea in the marine dogfish In contrast to teleosts, when changes in cell volume, hematocrit, and, therefore, we speculate that high levels of CS may also play an hemoglobin concentration and hemoglobin oxygen affinity are ob- important role in osmoregulation in freshwater stingrays. Potamotrygon served under stress conditions (Nikinmaa, 1990; Wendelaar Bonga, sp. from the ion poor Rio Negro exhibited plasma ion levels typical 1997), in some elasmobranch species erythrocytes do not respond to of freshwater teleosts and ammonia is the main nitrogen waste (Wood catecholamine release (Lowe et al., 1995). Consequently there are no et al., 2002). In the present study ammonia excretion rates, basal plasma measurable changes in red cell swelling and hematocrit (Brill et al., Cl−, urea and glucose levels were similar to values found by Wood et al. 2008), and loading of erythrocytes from hemopoietic tissues (Opdyke (2002), but plasma osmolarity and hematocrit were higher than the and Opdyke, 1971) are observed. In some cases the erythrocyte

Table 2 Blood variables obtained for the cururu stingrays during the transport experiment. No signiﬁcant differences were observed.

Treatments Htc RBC MCV Glucose Urea (%) (106 μL−1) (fL) (mmol L−1) (mmol L−1)

Basal 22.61±0.89 0.39±0.03 588.1±39.13 1.65±0.06 0.15±0.03 Before transport 22.50±0.71 0.32±0.04 713.0±52.25 1.53±0.30 0.42±0.14 Control 3 h 21.92±0.99 0.30±0.02 725.4±43.14 1.94±0.22 0.21±0.05 Antibiotic 3 h 20.16±0.88 0.34±0.02 607.7±37.00 2.07±0.14 0.21±0.03 Probiotic 3 h 18.92±1.01 0.33±0.03 571.5±44.00 1.88±0.16 0.15±0.03 Control 12 h 18.50±1.02 0.28±0.01 642.6±30.55 1.71±0.21 0.28±0.03 Antibiotic 12 h 18.42±0.89 0.30±0.02 628.5±53.37 1.88±0.19 0.68±0.29 Probiotic 12 h 22.66±0.40 0.32±0.02 713.9±45.94 1.70±0.28 0.48±0.13 Control 24 h 19.67±1.58 0.30±0.02 650.1±52.82 1.25±0.22 0.11±0.02 Antibiotic 24 h 21.66±1.33 0.30±0.02 722.7±60.78 1.65±0.20 0.11±0.01 Probiotic 24 h 21.66±0.51 0.30±0.02 724.9±43.87 1.97±0.15 0.13±0.01 Author's personal copy

144 R.P. Brinn et al. / Comparative Biochemistry and Physiology, Part A 162 (2012) 139–145 swelling, instead of adrenergic action, is due to changes in body volume Carvalho, M.R., Lovejoy, N.N., Rosa, R.S., 2003. Family Potamotrygonidae (river fl − stingrays). In: Reis, R.E., Kullander, S.O., Ferraris Jr., C.J. (Eds.), Check List of the and net in ux of Cl (and water), since red cell membranes in elas- Freshwater Fishes of South and Central America. : Porto Alegre. Editora da + + mobranchs are more permeable to this ion than Na and K (Anderson Pontifícia Universidade Católica. Porto Alegre, pp. 22–29. et al., 2007; Brill et al., 2008). However, in the Amazonian stingray, Chao, N.L., Petry, P., Prang, G., Sonneschien, L., Tlusty, M., 2001. Conservation and Management of Ornamental Fish Resources of the Rio Negro Basin, Amazonia. transport conditions did not elicit an increase in red cell volume, Brazil - Project Piaba, EDUA, Manaus. 309 p. estimated by MCV values. The intensity of the observed changes in the Charvet-Almeida, P., Araujo, M.L.G., Almeida, M.P., 2005. Reproductive aspects of water quality parameters, i.e., increases in total ammonia levels, pH and freshwater stingrays (Chondrichthyes: Potamotrygonidae) in the Brazilian Amazon – electric conductivity, and a concomitant decrease in dissolved oxygen basin. J. Northw. Atl. Fish. Sci. 35, 165 171. Eddy, F.B., 1981. Effects of stress on osmotic and ionic regulation in fish. In: Pickering, −1 levels (~2.0 mg L ), were not sufficient to promote adjustments in the A.D. (Ed.), Stress and fish. Academic Press, pp. 77–102. hematologic variables. Frick, L.H., Reina, R.D., Walker, T.I., 2010. Stress related physiological changes and post- release survival of Port Jackson sharks (Heterodontus portusjacksoni) and gummy sharks (Mustelus antarcticus) following gill-net and longline capture in captivity. 5. Conclusions Exp. Mar. Biol. Ecol. 385, 29–37. Gomes, L.C., Brinn, R.P., Marcon, J.L., Dantas, L.A., Brandao, F.R., de Abreu, J.S., McComb, D.M., Baldisserotto, B., 2008. Using Efinol (R) L during transportation of marbled We can conclude that corticosterone can be used as an indicator of hatchetfish, Carnegiella strigata (Gunther). Aquacult. Res. 39, 1292–1298. stress for this species of Amazonian stingray which do not have a Gomes, L.C., Brinn, R.P., Marcon, J.L., Dantas, L.A., Brandao, F.R., de Abreu, J.S., Lemos, fi typical teleost hyperglycemic reaction to stress. Their osmoregulatory P.E.M., McComb, D.M., Baldisserotto, B., 2009. Bene ts of using the probiotic Efinol(R)L during transportation of cardinal tetra, Paracheirodon axelrodi (Schultz), response is similar to most freshwater teleosts. The use of probiotic in the Amazon. Aquacult. Res. 40, 157–165. and antibiotic as water additives during transport did not reduce stress Gonzalez, R.J., Wood, C.M., Wilson, R.W., Patrick, M.L., Bergman, H.L., Harahara, A., Val, fi related responses and should be avoided. A.L., 1998. Effects of water pH and calcium concentration on ion balance in sh of the Rio Negro. Amazon. Physiol. Zool. 71, 15–22. Hazon, N., Henderson, I.W., 1984. Secretory dynamics of 1α-Hydroxycorticosterone in fi – Acknowledgements the elasmobranch sh, Scyliorhinus canicula. J. Endocrinol. 103, 205 211. Heinicke, M.P., Naylor, G.J.P., Hedges, S.B., 2009. Cartilaginous fishes (Chonrichthyes). In: Hedges, S.B., Kumar, S. (Eds.), The Time Tree of Life. Oxford University Press, The results obtained in this manuscript were possible due to New York, pp. 320–327. Idler, D.R., Truscott, B., 1966. 1α-Hydroxycorticosterone from cartilaginous fish: a new funding by the National Geographic Society conservation fund (no. – fi adrenal steroid in blood. J. Fisher. Res. Bd. Canada 23, 615 619. C47-04). The Scienti c expedition as well as the collection of wild Idler, D.R., Truscott, B., 1967. 1α-Hydroxycorticosterone: synthesis in vitro and stingrays for analysis was previously authorized by the Brazilian properties of an interrenal steroid in the blood of cartilaginous fish (Genus Raja). Ministry of Science and Technology (MCT/CNPq, process no. EXC Steroids 9, 457–477. Idler, D.R., Truscott, B., 1968. The widespread occurrence of a corticosteroid 1α- 023/05 to J. L. Marcon), and by the Federal Environmental Agency hydroxilase in the interrenal of elasmobranchii. J. Endocrinol. 40, 515–526. (process no. 098/2005 DIFAP/IBAMA-DF). BB, JLM and LCG are Kime, D.E., 1977. Measurement of 1α-hydroxycorticosterone and other corticosteroids research fellowship recipients of CNPq/Brazil. The authors gratefully in elasmobranch plasma by radioimmunoassay. Gen. Comp. Endocrinol. 33, 344–351. acknowledge AES, the Fisheries Conservation Foundation, and Save Lowe, T.E., Wells, R.M.G., Baldwin, J., 1995. Absence of regulated blood-oxygen our Seas Foundation for their support. transport in response to strenuous exercise by the shovelnosed ray, Rhinobatos typus. Mar. Freshw. Res. 46, 441–446. Manire, C.A., Rasmussen, L.E.L., Maruska, K.P., Tricas, T.C., 2007. Sex, seasonal, and References stress-related variations in elasmobranch corticosterone concentrations. Comp. Biochem. Physiol. A148, 926–935. American Public Health Association, American Water Works Association, Water Martin, R.A., 2005. Conservation of freshwater and euryhaline elasmobranchs: a review. Environment Federation, 1992. Standard Methods for the Examination of Water J. Mar. Biol. Assoc. U. K. 85, 1049–1073. and Wastewater, 18th ed. American Public Health Association, NewYork, NY, USA. Matsuo, A.Y.O., Val, A.L., 2003. Fish adaptations to Amazonian blackwaters. In: Val, A.L., Anderson, W.G., Taylor, J.R., Good, J.P., Hazon, N., Grosell, M., 2007. Body fluid volume Kapoor, B.G. (Eds.), Fish Adaptations. Science Publishers, Inc, Enfield. regulation in elasmobranch fish. Comp. Biochem. Physiol. A 148, 3–13. Mazeaud, M.M., Mazeaud, F., 1981. Adrenergic responses to stress in fish. In: Pickering, Armour, K.J., O'Toole, L.B., Hazon, N., 1993. Mechanisms of ACTH- and angiotensin II- A.D. (Ed.), Stress and Fish. Academic Press, pp. 49–76. stimulated 1α-hydroxycorticosterone secretion in the dogfish, Scyliorhinus McDonald, G., Milligan, L., 1997. Ionic, osmotic and acid-base regulation in stress. In: canicula. J. Mol. Endocrinol. 10, 235–244. Iwama, G.W., Pickering, A.D., Sumpter, J.P., Schreck, C.B. (Eds.), Fish Stress and Arnold, J.E., 2005. Hematology of the sandbar shark, Carcharhinus plumbeus: Health in Aquaculture. University Press, Cambridge, pp. 119–144. standardization of complete blood count techniques for elasmobranchs. Vet. Clin. McGowan, D.W., Kajiura, S.M., 2009. Eletroreception in the euryhaline stingray, Pathol. 115–123. Dasyatis sabina. J. Exp. Biol. 212, 1544–1552. Ballantyne, J.S., 1997. Jaws: the inside story. The metabolism of elasmobranch fishes. Morgan, J.D., Iwama, G.K., 1996. Cortisol-induced changes in oxygen consumption and Comp. Biochem. Physiol. B 118, 703–742. ionic regulation in coastal cutthroat trout (Oncorhynchus clarki clarki) parr. Fish Benfey, T.J., Biron, M., 2000. Acute stress response in triploid rainbow trout Physiol. Biochem. 15, 385–394. (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis). Aquaculture 184, Morgan, J.D., Iwama, G.K., 1997. Measurement of stressed states in the field. In: Iwama, 167–176. G.K., Pickering, A.D., Summer, J.P., Schreck, C.B. (Eds.), Fish Stress and Health in Bittner, A., Lang, S., 1980. Some aspects of the osmoregulation of Amazonian freshwater Aquaculture. Cambridge University Press, Cambridge, pp. 247–270. stingrays (Potamotrygon hystrix)—I. Serum osmolality, sodium and chloride Mortatti, J., Probst, J.L., 2003. Silicate rock weathering and atmospheric/soil CO2 uptake content, water content, hematocrit and urea level. Comp. Biochem. Physiol. A 67, in the Amazon basin estimated from river water geochemistry: seasonal and spatial 9–13. variations. Chem. Geol. 197, 177–196. Bridgham, J.T., Carroll, S.M., Thorton, J.W., 2006. Evolution of hormone-receptor Nikinmaa, M., 1990. Vertebrate Red Blood Cells: Adaptations of Function to Respiratory complexity by molecular exploitation. Science 312 (5770), 97–101. Requirements. Springer-Verlag, Berlin. 262p. Brill, R., Bushnell, P., Schroff, S., Seifert, R., Galvin, M., 2008. Effects of anaerobic exercise O'Toole, L.B., Armour, K.J., Decourt, C., Hazon, N., Lahlou, B., Henderson, I.W., 1990. accompanying catch-and-release fishing on blood-oxygen affinity of the sandbar Secretory patterns of 1α-hydroxycorticosterone in the isolated perifusedinterrenal shark (Carcharhinus plumbeus, Nardo). J. Exp. Mar. Biol. Ecol. 354, 132–143. gland of the dogfish. Scyliorhinuscanicula. J. Mol. Endocrinol. 5, 55–60. Carlson, J.K., Parsons, G.R., 2003. Respiratory and hematological responses of the Opdyke, D.F., Opdyke, N.E., 1971. Splenic responses to stimulation in Squalus acanthias. bonnethead shark, Sphyrna tiburo, to acute changes in dissolved oxygen. Exp. Mar. Am. J. Physiol. 221, 623–625. Biol. Ecol. 294, 15–26. Piermarini, P.M., Evans, D.H., 1998. Osmoregulation of the Atlantic stingray (Dasyatis Carroll, S.M., Bridgham, J.T., Thornton, J.W., 2008. Evolution of hormone signaling in sabina) from the freshwater Lake Jesup of the St. Johns River. Florida. Physiol. Zool. elasmobranchs by exploitation of promiscuous receptors. Mol. Biol. Evol. 25, 71, 553–560. 2643–2652. Raschi, W., Mackanos, L.A., 1989. The structure of the ampullae of lorenzini in dasyatis Carvalho, M.R., Lovejoy, N.R., 2011. Morphology and phylogenetic relationships of a garouaensis and its implications on the evolution of freshwater electroreceptive remarkable new genus and two new species of Neotropical freshwater stingrays systems. J. Exp. Zool. 101–111. from the Amazon basin (Chondrichthyes: Potamotrygonidae). Zootaxa 2776, Rasmussen, L.E.L., Crow, G.L., 1993. Serum corticosterone concentrations in immature 13–48. captive whitetip reef sharks, Triaenodon obesus. J. Exp. Zool. 267, 283–287. Carvalho, M.R., Ragno, M.P., 2011. An unusual, dwarf new species of Neotropical Rasmussen, L.E.L., Gruber, S.H., 1990. Serum levels of circulating steroid hormones in freshwater stingray, Plesiotrygon nana sp. nov., from the upper and mid Amazon free-ranging carcharhinoid sharks. In: Pratt, H.L., Taniuchi, T., Gruber, S.H. (Eds.), basin: the second species of Plesiotrygon (Chondrichthyes: Potamotrygonidae). Elasmobranchs as Living Resources, NOAA Technical Report-NMFS, Seattle, pp. Papeis Avulsos de Zoologia 51 (7), 101–138. 143–155. Author's personal copy

R.P. Brinn et al. / Comparative Biochemistry and Physiology, Part A 162 (2012) 139–145 145

Rasmussen, L.E.L., Gruber, S.H., 1993. Serum concentration of reproductively-related Thorson, T.B., Langhammer, J.K., Oetinger, M.I., 1983. Reproduction and development of circulating steroid hormones in the free-ranging lemon shark, Negaprion brevirotris. the South American freshwater stingrays. Potamotrygon circularis and P. motoro. Envi. Biol. Fishes 38, 167–174. Environ. Biol. Fish. 9, 3–24. Ricart-Jane´, D., Rodriguez-Sureda, V., Benavides, A., Peinado-Onsurbe, J., Lopez-Tejero, Truscott, B., Idler, D.R., 1968. The widespread occurrence of a corticosteroid 1α M.D., Llobera, M., 2002. Immobilization stress alters intermediate metabolism and hydrolase in the interrenals of Elamobranchii. J. Endocrinol. 40, 515–526. circulating lipoproteins in the rat. Metabolism 51, 925–931. Truscott, B., Idler, D.R., 1972. Corticosteroids in plasma of elasmobranchs. Comp. Snelson, F., Rasmussen, L., Johnson, M., Hess, D., 1997. Serum concentrations of steroid Biochem. Physiol. A 42, 41–50. hormones during reproduction in the Atlantic stingray, Dasyatis sabina. Gen. Comp. Verdouw, H., Vanechted, C.J.A., Dekkers, E.M.J., 1978. Ammonia determination based on Endocrinol. 108, 67–79. indophenol with sodium salicylate. Water Res. 12, 399–402. Speer- Roesch, B., Ip, Y.K., Ballantyne, J.S., 2006. Metabolic organization of freshwater, Wendelaar Bonga, S.E., 1997. The stress response in fish. Physiol. Revs. 77, 591–625. euryhaline, and marine elasmobranchs: implications for the evolution of energy Wilhelm Filho, D., Eble, G.J., Kassner, G., Caprario, F.X., Dafret, A.L., Ohira, M., 1992. metabolism in sharks and rays. J. Exp. Biol. 206, 2495–2508. Comparative hematology in marine fish. Comp. Biochem. Physiol. A102, 311–321. Tavares-Dias, M., Moraes, F.R., 2004. Hematologia de peixes teleósteos. Villimpress Wojtaszek, J., Dziewulska-Szwajkowska, D., Lozinska-Gabska, M., Adamowicz, A., Complexo Gráfico. . 144p. Dzugaj, A., 2002. Hematological effects of high dose of cortisol on the carp (Cyprinus Teague, C.R., Dhabhar, F.S., Barton, R.H., Beckwith-Hall, B., Powell, J., Cobain, M., Singer, carpio L.): cortisol effect on the carp blood. Gen. Comp. Endocrinol. 125, 176–183. B., McEwen, B., Lindon, J., Nicholson, J., Holmes, H., 2007. Metabonomic studies on Wood, C.M., Matsuo, A.Y.O., Gonzalez, R.J., Wilson, R.W., Patrick, M.L., Val, A.L., 2002. the physiological effects of acute and chronic psychological stress in Sprague– Mechanisms of ion transport in Potamotrygon, a stenohaline freshwater elasmo- Dawley rats. J. Proteome Res. 6, 2080–2093. branch native to the ion-poor blackwaters of the Rio Negro. J. Exp. Biol. 205, Thornton, J.W., 2001. Evolution of vertebrate steroid receptors from an ancestral 3039–3054. estrogen receptor by ligand exploitation and serial genome expansions. Proc. Natl. Zall, D.M., Fisher, M.D., Garner, Q.M., 1956. Photometric determination of chlorides in Acad. Sci. USA 98, 5671–5676. water. Anal. Chem. 28, 1665–1678.