Cytogenetic and molecular characteristics of Potamotrygon motoro and Potamotrygon sp. (Chondrichthyes,Myliobatiformes, Potamotrygonidae) from the Amazon basin: Implications for the taxonomy of the genus

The chromosomes of two freshwater stingrays, Potamotrygon motoro and Potamotrygon sp., from the Amazon River basin in Brazil were investigated using integrated molecular (cytochrome c oxidase subunit 1) and cytogenetic analyses. Potamotrygon motoro presented intraspecific variation in the diploid number, with 2n=66 in the females and 2n=65 in the males, while Potamotrygon sp. had a karyotype with 66 chromosomes, in both sexes. The C-banding revealed the presence of heterochromatic blocks accumulated in the centromeric region of all the chromosomes in both species. The FISH assays with 18S DNA probes highlighted the terminal region of three or four chromosome pairs in P. motoro and seven chromosomes in Potamotrygon sp. The rDNA 5S sequences were found in only one chromosomal pair in both species. The interspecific genetic distance based on the COI sequences, between P. motoro and Potamotrygon sp. from Amazon River was 10.8%, while that between the Amazonian P. motoro and Potamotrygon amandae from the Paraná River was 2.2%, and the genetic distance between Potamotrygon sp. and P. amandae was 11.8%. In addition to the new insights on the cytogenetics of the study species, the results of the present study confirmed the existence of heteromorphic sex-linked chromosomes in P. motoro.     

Stingers in a strange land: South American freshwater stingrays (Potamotrygonidae) in Singapore

South American freshwater stingrays of the family Potamotrygonidae have long been popular worldwide as ornamentals in the aquarium trade. Despite this, there have so far not been any reported cases of their establishment elsewhere in the world. Here, we document the ocellate river stingray, Potamotrygon motoro, as having established itself in Singapore. This represents the first alien record of a South American freshwater stingray outside the Neotropics.     

Chromosomes of three freshwater stingrays (Rajiformes Potamotrygonidae) from the Rio Negro basin, Amazon, Brazil

Potamotrygonidae is the representative family of South American freshwater elasmobranchs. It is a monophyletic group containing 20 species grouped into three genera. Three species belonging to two genera of this family were collected from the middle Negro River, Amazonas, Brazil, and studied cytogenetically: Paratrygon aiereba, Potamotrygon motoro and Potamotrygon orbignyi. Paratrygon aiereba presented 2n = 90 chromosomes and 4M+2SM+10ST+74A. Both species of Potamotrygon presented 2n = 66 chromosomes and differed in their chromosomal formulas: P. motoro had 18M+12SM+10ST+26A and P. orbignyi had 22M+10SM+8ST+26A. No sex heteromorphism was detected. The Fundamental Number (FN) was 106 for the three species. A system of multiple NORs was found in the three species, but with interspecific differences in terms of location and position of the active Ag-NORs sites. Paratrygon aiereba presented only four sites on the short arms of two chromosomal pairs, both in terminal regions. Potamotrygon motoro presented seven sites, on the long and short arms, all in terminal regions of non-homologous chromosomes; P. orbignyi presented eight sites on the long arms, all in terminal regions, of non-homologous chromosomes. The constitutive heterochromatin was in pericentromeric regions of all chromosomes, and no significant interspecific difference was found in relation to this marker.     

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

Potamotrygon cf. histrix (cururu stingray) are endemic freshwater stingrays from the middle region of the Rio Negro in the Brazilian Amazon basin and are exported worldwide as ornamentals caught by artisanal fishermen. The transport process from capture to final destination is long and stressful. This study quantified stress related changes in corticosterone, blood and water samples (baseline, pre-transport, 3h, 12h and 24h) analyzed during a transport experiment which tested two water additives (tetracycline and the probiotic Efinol). There was a significant stepwise increase in corticosterone levels in stingrays over transport time in combination with osmoregulatory disturbances suggesting a stress related role of this corticosteroid. There were significant increases in water conductivity, Na(+) and K(+) losses and ammonia excretion. Blood parameters such as glucose, hematocrit, red blood count and urea did not change significantly during the experiment. Glucose levels did not increase significantly during transport and this may be due to the fact that 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.     

Reproduction and development of the South American freshwater stingrays,Potamotrygon circularis and P. motoro

Synopsis Observations of reproductive features and body measurements were made on wild-caught, freshwater stingrays, Potamotrygon circularis and P. motoro, from the Amazon drainage of western Brazil and southern Colombia. Further observations were made in Detroit's Belle Isle Aquarium on a captive pair of P. motoro and their descendants, which constitute the first known captive breeding colony of potamotrygonids. The gross structure and function of female and male reproductive systems are described. There is no obvious difference between those of the two species. They are aplacentally viviparous, the young being nourished in advanced stages by uterine milk secreted by trophonemata. Size at onset and completion of sexual maturation, breeding season and behavior, gestation period, litter size and sex ratios are discussed. Up to 21 proportional measurements were made on several fetal and postnatal stages of both species. Several proportional changes occur in very early fetal life, but most body proportions undergo only minor changes from advanced fetal through adult stages. A growth curve is proposed for P. motoro based on observations of the captive colony.     

Identification and validation of single nucleotide polymorphisms as tools to detect hybridization and population structure in freshwater stingrays

Single nucleotide polymorphism (SNP) markers were identified and validated for two stingrays species, Potamotrygon motoro and Potamotrygon falkneri, using double digest restriction‐site associated DNA (ddRAD) reads using 454‐Roche technology. A total of 226 774 reads (65.5 Mb) were obtained (mean read length 289 ± 183 bp) detecting a total of 5399 contigs (mean contig length: 396 ± 91 bp). Mining this data set, a panel of 143 in silico SNPs was selected. Eighty‐two of these SNPs were successfully validated and 61 were polymorphic: 14 in P. falkneri, 21 in P. motoro, 3 in both species and 26 fixed for alternative variants in both species, thus being useful for population analyses and hybrid detection.     

The ocellate river stingray (Potamotrygon motoro) exploits vortices of sediment to bury into the substrate

Particle image velocimetry and video analysis were employed to determine the pectoral-fin mechanism used by the stingray Potamotrygon motoro to bury into sand. Rapid oscillations of the body and folding motions of the posterior portion of the pectoral fin suspended sediment beneath the pectoral disc and directed vortices of sediment onto the dorsal surface, where they dissipated and the sediment settled. Body coverage was increased by increased fin displacement and speed and also by the occasional collision of vortices that redirected sediment flow towards the head and tail.     

Effects of biological traits on capture-induced parturition in a freshwater stingray and perspectives for species management

Elasmobranchs are particularly vulnerable to overexploitation and population depletion, especially due to their life-history traits, such as low reproductive output and slow growth. Given that capture-induced parturition (abortion or premature birth) is a common consequence of fisheries in elasmobranchs, but still little studied, we investigated how the abortion/premature birth process varies in response to reproductive traits in a freshwater stingray, Potamotrygon amandae. Our results revealed that capture-induced parturition was affected by reproductive traits, such as litter size (one to seven) and gestation stage. The event occurred faster in pregnant females with high litter size during late pregnancy. Also, as found in other elasmobranchs, litter size was positively correlated with maternal size. These findings indicate that larger pregnant females in late pregnancy are more vulnerable to capture-induced parturition. This study improves our understanding of the capture-induced parturition process in stingrays, and provides useful information for management strategies and future recommendations for elasmobranch conservation.     

Spatial memory and orientation strategies in the elasmobranch<Emphasis Type="Italic"> Potamotrygon motoro</Emphasis>

We investigated whether juvenile freshwater stingrays (Potamotrygon motoro) can solve spatial tasks by constructing a cognitive map of their environment. Two experimental conditions were run: allocentric and ego-allocentric. Rays were trained to locate food within a four-arm maze placed in a room with visual spatial cues. The feeding location (goal) within the maze (room) remained constant while the starting position varied for the allocentrically but not for the ego-allocentrically trained group. After training, all rays solved the experimental tasks; however, different orientation strategies were used within and between groups. Allocentrically trained rays reached the goal via novel routes starting from unfamiliar locations, while ego-allocentrically trained rays primarily solved the task on the basis of an egocentric turn response. Our data suggest that P. motoro orients by constructing a visual cognitive map of its environment, but also uses egocentric and/or other orientation strategies alone or in combination for spatial orientation, a choice which may be governed by the complexity of the problem. We conclude that spatial memory functions are a general feature of the vertebrate brain.     

Morphological and histochemical characterization of the pectoral fin muscle of batoids

Batoids differ from other elasmobranch fishes in that they possess dorsoventrally flattened bodies with enlarged muscled pectoral fins. Most batoids also swim using either of two modes of locomotion: undulation or oscillation of the pectoral fins. In other elasmobranchs (e.g., sharks), the main locomotory muscle is located in the axial myotome; in contrast, the main locomotory muscle in batoids is found in the enlarged pectoral fins. The pectoral fin muscles of sharks have a simple structure, confined to the base of the fin; however, little to no data are available on the more complex musculature within the pectoral fins of batoids. Understanding the types of fibers and their arrangement within the pectoral fins may elucidate how batoid fishes are able to utilize such unique swimming modes. In the present study, histochemical methods including succinate dehydrogenase (SDH) and immunofluoresence were used to determine the different fiber types comprising these muscles in three batoid species: Atlantic stingray (Dasyatis sabina), ocellate river stingray (Potamotrygon motoro) and cownose ray (Rhinoptera bonasus). All three species had muscles comprised of two muscle fiber types (slow-red and fast-white). The undulatory species, D. sabina and P. motoro, had a larger proportion of fast-white muscle fibers compared to the oscillatory species, R. bonasus. The muscle fiber sizes were similar between each species, though generally smaller compared to the axial musculature in other elasmobranch fishes. These results suggest that batoid locomotion can be distinguished using muscle fiber type proportions. Undulatory species are more benthic with fast-white fibers allowing them to contract their muscles quickly, as a possible means of escape from potential predators. Oscillatory species are pelagic and are known to migrate long distances with muscles using slow-red fibers to aid in sustained swimming.     

Characterization of cDNAs encoding cholesterol side chain cleavage and 3beta-hydroxysteroid dehydrogenase in the freshwater stingray Potamotrygon motoro

The interrenal gland (adrenocortical homolog) of elasmobranchs produces a unique steroid, 1α-hydroxycorticosterone (1α-B). The synthesis of this and most other steroids requires both cholesterol side chain cleavage (CYP11A) and 3β-hydroxysteroid dehydrogenase (HSD3). To facilitate the study of elasmobranch steroidogenesis, we isolated complementary DNAs encoding CYP11A and HSD3 from the freshwater stingray Potamotrygon motoro. The P. motoro CYP11A (2182 bp total length) and HSD3 (2248 bp total length) cDNAs harbor open reading frames that encode proteins of 542 and 376 amino acids (respectively) that are similar (CYP11A: 39–61% identical; HSD3: 36–53% identical) to their homologs from other vertebrates. In molecular phylogenetic analysis, P. motoro CYP11A segregates with CYP11A proteins (and not with related CYP11B proteins) and P. motoro HSD3 segregates with steroidogenic HSD3 proteins from other fishes. CYP11A and HSD3 mRNA is found only in interrenal and gonadal tissues, indicating de novo steroidogenesis is restricted to these tissues. Because 1α-B is thought to act in the elasmobranch response to hydromineral disturbances, we examined the effect of adapting P. motoro to 10 ppt seawater on mRNAs encoding steroidogenic genes. The P. motoro response to this salinity challenge does not include interrenal hypertrophy or an increase in the levels of interrenal CYP11A, HSD3 or steroidogenic acute regulatory protein (StAR) mRNA. This study is the first to isolate full length cDNAs encoding elasmobranch CYP11A and HSD3 and the first to examine the regulation of steroidogenic genes in elasmobranch interrenal cells.     

Potamotrygon marquesi,a new species of neotropical freshwater stingray (Potamotrygonidae) from the Brazilian Amazon Basin

Potamotrygon marquesi, sp. nov., is described and compared with other species of Potamotrygon occurring in the Amazon Basin. The identity of this new species is supported by an extensive external and internal morphological study including coloration pattern, squamation, skeleton and ventral lateral-line canals. Morphometrics and meristics were used to further distinguish P. marquesi from congeners. Potamotrygon marquesi was first considered to fall within the range of variation found in P. motoro. However, even with an extensive variation in coloration observed in P. motoro, this new species presents a series of autapomorphies that confidently distinguishes it from what is understood as the morphological variation found in P. motoro. Additional morphological characters that diagnose P. marquesi include three angular cartilages, asymmetrical star-shaped denticles, a single regular row of spines on tail dorsum, lateral row of caudal spines near the sting insertion, dorsal disc background in beige and grey mixed with shades of grey and bearing open and closed bicolored rings, among others. Although presenting a gap of distribution along the west–east extension of the Amazon Basin, its diagnostic charactistics are consistent in both recorded regions. Our study supports the need for many morphological characters to robustly distinguish members of Potamotrygoninae considering their extremely variable dorsal disc color pattern.     

Diet Reconstruction and Resource Partitioning of a Caribbean Marine Mesopredator Using Stable Isotope Bayesian Modelling

The trophic ecology of epibenthic mesopredators is not well understood in terms of prey partitioning with sympatric elasmobranchs or their effects on prey communities, yet the importance of omnivores in community trophic dynamics is being increasingly realised. This study used stable isotope analysis of ¹⁵N and ¹³C to model diet composition of wild southern stingrays Dasyatis americana and compare trophic niche space to nurse sharks Ginglymostoma cirratum and Caribbean reef sharks Carcharhinus perezi on Glovers Reef Atoll, Belize. Bayesian stable isotope mixing models were used to investigate prey choice as well as viable Diet-Tissue Discrimination Factors for use with stingrays. Stingray δ¹⁵N values showed the greatest variation and a positive relationship with size, with an isotopic niche width approximately twice that of sympatric species. Shark species exhibited comparatively restricted δ¹⁵N values and greater δ¹³C variation, with very little overlap of stingray niche space. Mixing models suggest bivalves and annelids are proportionally more important prey in the stingray diet than crustaceans and teleosts at Glovers Reef, in contrast to all but one published diet study using stomach contents from other locations. Incorporating gut contents information from the literature, we suggest diet-tissue discrimination factors values of Δ¹⁵N ≊ 2.7‰ and Δ¹³C ≊ 0.9‰ for stingrays in the absence of validation experiments. The wide trophic niche and lower trophic level exhibited by stingrays compared to sympatric sharks supports their putative role as important base stabilisers in benthic systems, with the potential to absorb trophic perturbations through numerous opportunistic prey interactions.     

Using acoustic telemetry to assess patterns in the seasonal residency of the Atlantic stingray <Emphasis Type="Italic">Dasyatis sabina</Emphasis>

The Atlantic stingray Dasyatis sabina is found along the Atlantic and Gulf coasts of the United States. It consumes commercially important shrimp and crabs, and its diet overlaps with recreationally valuable red drum Sciaenops ocellatus and pompano Trachinotus spp. Despite the potential economic impact of this species, it is unclear whether the Atlantic stingray is present year-round or only seasonally in coastal habitats. The objective of this study was to assess the seasonal residency patterns of Atlantic stingrays in two creek systems in the Savannah River estuary. Forty stingrays were tracked using acoustic telemetry, and a seasonal residence index was calculated for each individual. Atlantic stingrays were present year-round in the Savannah River estuary, as 15 % (n = 6) of the tagged rays remained in the study areas throughout the year. This is the northernmost region where this species has been documented to be present all year. Of the 85 % (n = 34) of the rays that migrated during winter, 38 % (n = 13) of those were detected within the estuary less than 20 km away. Had trawls or mark-recapture been used instead, the few animals remaining in the creek systems and/or those that migrated may not have been collected. Acoustic telemetry is a more accurate means of studying the residency of fishes than periodic sampling that requires capture, and researchers should consider incorporating this technology in future studies about fish-environment interactions. Underestimating the presence of a species could result in miscalculation of its economic and ecological impact and, by extension, result in the implementation of ineffective or even detrimental management strategies.     

Trophic ecology and metabolism of two species of nonnative freshwater stingray (Chondrichthyes: Potamotrygonidae)

The stingrays Potamotrygon amandae and Potamotrygon falkneri are nonnative species established in the Upper Paraná basin. Although they are widely distributed, few studies on their diets or respective metabolic responses exist. Therefore, the aim was to evaluate the dietary composition, trophic niche breadth and lipid/protein concentrations in muscle and hepatic tissues of these two species, as well as the interrelationships between them. The individuals were collected in two areas on the Upper Paraná River. The stomachs and samples of muscle and liver tissues were taken for analysis. A broad dietary spectrum was observed for both species, along with differences in dietary composition, with a higher consumption of detritus by P. amandae and Baetidae by P. falkneri. No differences were observed in the trophic niche breadth. Regarding the metabolic variables, differences were only found in the hepatic protein, with a higher content observed in P. falkneri. A significant positive correlation was observed between items of animal origin and detritus with muscle protein for this species. This shows that such feeding habits, which are characteristic of a generalist, influenced the metabolism of the species and possibly contributed to the successful adjustment of the species to new habitats in the Upper Paraná River.

     

Supplemental Feeding for Ecotourism Reverses Diel Activity and Alters Movement Patterns and Spatial Distribution of the Southern Stingray,Dasyatis americana

Southern stingrays, Dasyatis americana, have been provided supplemental food in ecotourism operations at Stingray City Sandbar (SCS), Grand Cayman since 1986, with this site becoming one of the world’s most famous and heavily visited marine wildlife interaction venues. Given expansion of marine wildlife interactive tourism worldwide, there are questions about the effects of such activities on the focal species and their ecosystems. We used a combination of acoustic telemetry and tag-recapture efforts to test the hypothesis that human-sourced supplemental feeding has altered stingray activity patterns and habitat use at SCS relative to wild animals at control sites. Secondarily, we also qualitatively estimated the population size of stingrays supporting this major ecotourism venue. Tag-recapture data indicated that a population of at least 164 stingrays, over 80% female, utilized the small area at SCS for prolonged periods of time. Examination of comparative movements of mature female stingrays at SCS and control sites revealed strong differences between the two groups: The fed animals demonstrated a notable inversion of diel activity, being constantly active during the day with little movement at night compared to the nocturnally active wild stingrays; The fed stingrays utilized significantly (p<0.05) smaller 24 hour activity spaces compared to wild conspecifics, staying in close proximity to the ecotourism site; Fed stingrays showed a high degree of overlap in their core activity spaces compared to wild stingrays which were largely solitary in the spaces utilized (72% vs. 3% overlap respectively). Supplemental feeding has strikingly altered movement behavior and spatial distribution of the stingrays, and generated an atypically high density of animals at SCS which could have downstream fitness costs for individuals and potentially broader ecosystem effects. These findings should help environmental managers plan mitigating measures for existing operations, and develop precautionary policies regarding proposed feeding sites.     

The study of the bioenergetic characteristics of the red blood cells of Black Sea fish: the common stingray (<Emphasis Type="Italic">Dasyatis pastinaca</Emphasis> L.) and black scorpionfish (<Emphasis Type="Italic">Scorpaena porcus</Emphasis> L.)

The oxygen consumption rate in red blood cell suspensions of two Black Sea fish species, a cartilaginous fish, the common stingray (Dasyatis pastinaca L.) and the teleost black scorpionfish (Scorpaena porcus L.) has been studied. The proposed stimulants of activators and inhibitors of the mitochondria electron transport chain had very predictable responses, indicating that mitochondria in fish erythrocytes have a classical set of respiratory enzymes. Despite the fact that the basic respiratory activity of common stingray erythrocytes was greater than those of the scorpionfish, the responses of common stingray red blood cells to the exposure during investigation of the respiratory activity of the mitochondria have an inverse relationship. The oxygen consumption rates in suspensions of scorpionfish erythrocytes in response to the stimulant were higher according to both the amplitude and the duration of the response. Investigations have shown the high energy potential of the red blood cell mitochondria of the scorpionfish and stingray. This may be the energy basis for maintaining the high intracellular concentrations of ATP required not only to keep an adequate level of intracellular metabolism, but also to provide a special mode of blood flow through the capillary beds.     

Linking local movement and molecular analysis to explore philopatry and population connectivity of the southern stingray Hypanus americanus

Limited data pertaining to life history and population connectivity of the data-deficient southern stingray (Hypanus americanus) are available. To determine potential vulnerabilities of their populations, this study aimed to analyse their movement patterns and genetic variability. A population of southern stingrays encompassing nine sites around Cape Eleuthera, the Bahamas, has been monitored using mark-recapture, spanning a 2.5 year period. Out of 200 individual stingrays, more than a third were encountered again. The home range of the females appears to be restricted, which supports the notion of high site residency. As resident populations of stingrays could suffer from a lack of population connectivity and be predestined for genetic isolation and local extirpation, this study further investigated the genetic connectivity of four sample sites in the central and western Bahamas. A haplotype analysis from the mitochondrial D-loop region showed that no distinct population structure strictly correlated with the sample site. These findings were complemented by five microsatellite loci that revealed high degrees in genotypic variability and little population differentiation. The results suggest gene flow mediated by both males and females.     

Observations of mesoscale movements in the short-tailed stingray, Dasyatis brevicaudata from New Zealand using a novel PSAT tag attachment method

Short-tailed stingrays (Dasyatis brevicaudata) are the largest stingrays in the world. They are common in New Zealand waters and aggregate seasonally in large numbers at the Poor Knights Islands Marine Reserve (PKMR), where they are a major attraction. Pop-up satellite archival tags (PSAT) were attached to two female short-tailed stingrays at two New Zealand offshore islands (within and nearby the PKMR) to investigate patterns of movement. These are the first PSAT data published for a stingray species. The rays were tagged for 62 and 151 days using a novel method of tag attachment developed specifically for ray species. Analyses of geolocation, depth and behavioural data indicate neither ray moved large distances (≤ 25 km) from the tagging locations. However, they showed a seasonal shift to deeper waters, progressively increasing time spent at greater depths and decreasing time spent at shallow depths towards winter. In addition, one ray displayed strong diel vertical movements. The method developed for PSAT attachment to stingrays proved successful and can be applied for long-term (> 6 months) movement and behavioural studies.     

Pendrin immunoreactivity in the gill epithelium of a euryhaline elasmobranch

Pendrin is an anion exchanger in the cortical collecting duct of the mammalian nephron that appears to mediate apical Cl(-)/HCO3(-) exchange in bicarbonate-secreting intercalated cells. The goals of this study were to determine 1) if pendrin immunoreactivity was present in the gills of a euryhaline elasmobranch (Atlantic stingray, Dasyatis sabina), and 2) if branchial pendrin immunoreactivity was influenced by environmental salinity. Immunoblots detected pendrin immunoreactivity in Atlantic stingray gills; pendrin immunoreactivity was greatest in freshwater stingrays compared with freshwater stingrays acclimated to seawater (seawater acclimated) and marine stingrays. Using immunohistochemistry, pendrin-positive cells were detected on both gill lamellae and interlamellar regions of freshwater stingrays but were more restricted to interlamellar regions in seawater-acclimated and marine stingray gills. Pendrin immunolabeling in freshwater stingray gills was more apical, discrete, and intense compared with seawater-acclimated and marine stingrays. Regardless of salinity, pendrin immunoreactivity occurred on the apical region of cells rich with basolateral vacuolar-proton-ATPase, and not in Na(+)-K(+)-ATPase-rich cells. We suggest that a pendrin-like transporter may contribute to apical Cl(-)/HCO3(-) exchange in gills of Atlantic stingrays from both freshwater and marine environments.