Metabolic organization of pregnant freshwater stingray and their offspring.

The elucidation of energetic patterns in adult viviparous elasmobranchs and their offspring can contribute to understanding ecophysiological questions, such as maternal-fetal metabolism and group life-history traits. We characterized the energetic substrates in pregnant individuals and stages of offspring development in the freshwater stingray Potamotrygon amandae. Our results show that the energetic distribution of the yolk is composed of more lipids than proteins, whereas the inverse pattern is observed in the egg and uterus, proving the plasticity of the energy provision of the species. As a novelty, we describe that yolk/intestine transfer occurs in this species.

Glycan expression as a tool for a deeper understanding of a reproductive gland in a skate of economic importance.

Atlantoraja platana is an oviparous skate endemic to the south-west Atlantic Ocean, and is one of the skate species most exploited by local industrial bottom trawl fisheries. Oviparous elasmobranchs encapsulate their eggs in complex egg cases produced by the oviductal gland (OG). This organ is exclusively present in these fishes and comprises four distinct zones: club, baffle, papillary and terminal. The relative size and structural complexity of these zones correlate with mode of reproduction. Glycans are known to play major roles in reproduction so their distribution in each zone of the OG could explain the functional multiplicity of the gland in skates, but this topic has not been previously investigated. In this study, morphological, histochemical and lectin-histochemical analysis revealed various novel aspects of A. platana's OG. The club, papillary and terminal zones positively stained for periodic acid Schiff's reagent (PAS) and Alcian Blue (AB), indicating the presence of neutral and acid mucopolysaccharides. However, the buffle zone was negative for PAS and AB stains, but was positive for all the lectins used. Each zone of the OG had a characteristic pattern of glycan expression. Finally, we confirmed the presence of sperm but not sperm storage. This is the first lectin-histochemical study of the OG in chondrichtyan fish and it has proven to be an important tool to understand some of the mechanisms of fertility and reproductive success in economic important species such as A. platana.

Novel signature fatty acid profile of the giant manta ray suggests reliance on an uncharacterised mesopelagic food source low in polyunsaturated fatty acids.

Traditionally, large planktivorous elasmobranchs have been thought to predominantly feed on surface zooplankton during daytime hours. However, the recent application of molecular methods to examine long-term assimilated diets, has revealed that these species likely gain the majority from deeper or demersal sources. Signature fatty acid analysis (FA) of muscle tissue was used to examine the assimilated diet of the giant manta ray Mobula birostris, and then compared with surface zooplankton that was collected during feeding and non-feeding events at two aggregation sites off mainland Ecuador. The FA profiles of M. birostris and surface zooplankton were markedly different apart from similar proportions of arachidonic acid, which suggests daytime surface zooplankton may comprise a small amount of dietary intake for M. birostris. The FA profile of M. birostris muscle was found to be depleted in polyunsaturated fatty acids, and instead comprised high proportions of 18:1ω9 isomers. While 18:1ω9 isomers are not explicitly considered dietary FAs, they are commonly found in high proportions in deep-sea organisms, including elasmobranch species. Overall, the FA profile of M. birostris suggests a diet that is mesopelagic in origin, but many mesopelagic zooplankton species also vertically migrate, staying deep during the day and moving to shallower waters at night. Here, signature FA analysis is unable to resolve the depth at which these putative dietary items were consumed and how availability of this prey may drive distribution and movements of this large filter-feeder.

Estimating tissue-specific discrimination factors and turnover rates of stable isotopes of nitrogen and carbon in the smallnose fanskate Sympterygia bonapartii (Rajidae).

This study aimed to estimate trophic discrimination factors (TDFs) and metabolic turnover rates of nitrogen and carbon stable isotopes in blood and muscle of the smallnose fanskate Sympterygia bonapartii by feeding six adult individuals, maintained in captivity, with a constant diet for 365 days. TDFs were estimated as the difference between δ(13) C or δ(15) N values of the food and the tissues of S. bonapartii after they had reached equilibrium with their diet. The duration of the experiment was enough to reach the equilibrium condition in blood for both elements (estimated time to reach 95% of turnover: C t95%blood = 150 days, N t95%blood = 290 days), whilst turnover rates could not be estimated for muscle because of variation among samples. Estimates of Δ(13) C and Δ(15) N values in blood and muscle using all individuals were Δ(13) Cblood = 1·7‰, Δ(13) Cmuscle = 1·3‰, Δ(15) Nblood = 2·5‰ and Δ(15) Nmuscle = 1·5‰, but there was evidence of differences of c.0·4‰ in the Δ(13) C values between sexes. The present values for TDFs and turnover rates constitute the first evidence for dietary switching in batoids based on long-term controlled feeding experiments. Overall, the results showed that S. bonapartii has relatively low turnover rates and isotopic measurements would not track seasonal movements adequately. The estimated Δ(13) C values in S. bonapartii blood and muscle were similar to previous estimations for elasmobranchs and to generally accepted values in bony fishes (Δ(13) C = 1·5‰). For Δ(15) N, the results were similar to published reports for blood but smaller than reports for muscle and notably smaller than the typical values used to estimate trophic position (Δ(15) N c. 3·4‰). Thus, trophic position estimations for elasmobranchs based on typical Δ(15) N values could lead to underestimates of actual trophic positions. Finally, the evidence of differences in TDFs between sexes reveals a need for more targeted research.

Analysis of food habits of skate Rioraja agassizii (Elasmobranchii, Rajidae) from southern Brazil / Análise quantitativa dos hábitos alimentares da raia Rioraja agassizii (Elasmobranchii, Rajidae) do Sudeste e Sul do Brasil

Abstract Catches and exports of skate Rioraja agassizii place this species as vulnerable to extinction on the IUCN Red List; therefore, biological and ecological knowledge becomes an important instrument for its conservation control. This study described and quantified the diet composition of R. agassizii by means of stomach analysis contents in the periods 2005-2006 and 2012-2013. We analyzed and quantified stomach contents in terms of abundance (%N), weight (%M), frequency of occurrence (% FO), and index of relative importance (IRI). The results showed differences in the food rates between the periods. However, the groups of food items were the same: Teleostei fish, decapods, and mollusks. In 2005-2006, the diet consisted mainly of shrimp, however, in 2012-2013 it consisted of fish, followed by decapods, especially shrimps. The differences in diets may be attributed to shrimp abundance, which do not characterize a change in the eating habits in 2012-2013, because, in addition to fish, shrimps were also important food sources. The presence of a certain prey is more related to its availability rather than the feeding preference of skate. The amount of ingested items is associated to biological and environmental factors, so that further studies relating diet with capture area, seasonality, depth, and other factors should be conducted.(AU)

Resumo A raia-santa, Rioraja agassizii é uma espécie endêmica da qual pouco se conhece sobre sua biologia e ecologia, o que a leva a ser classificada como vulnerável a extinção. O objetivo foi identificar e quantificar a composição da dieta alimentar da R. agassizii através da análise do conteúdo estomacal de exemplares capturados nos períodos de 2005-2006 e 2012-2013. Foram analisados os conteúdos alimentares e quantificados em abundância (N%), peso (M%), frequência de ocorrência (FO%) e índice de importância relativa (IRI) de cada item. Os resultados mostraram que em 2012-13 a dieta foi composta por peixes, seguido de decápodes, especialmente camarões, entretanto em 2005-06 foi composta basicamente por camarões. A diferença nas dietas não quer dizer que os hábitos alimentares mudaram, pois em 2012-13 além dos peixes, os camarões também foram classificados como maior importância alimentar. Considerando que a quantidade e variedade dos itens ingeridos está relacionada, principalmente, a fatores ambientais são necessários mais estudos que relacionem áreas de captura, sazonalidade e profundidade.(AU)

Venom gland transcriptome analyses of two freshwater stingrays (Myliobatiformes: Potamotrygonidae) from Brazil.

Stingrays commonly cause human envenoming related accidents in populations of the sea, near rivers and lakes. Transcriptomic profiles have been used to elucidate components of animal venom, since they are capable of providing molecular information on the biology of the animal and could have biomedical applications. In this study, we elucidated the transcriptomic profile of the venom glands from two different freshwater stingray species that are endemic to the Paraná-Paraguay basin in Brazil, Potamotrygon amandae and Potamotrygon falkneri. Using RNA-Seq, we identified species-specific transcripts and overlapping proteins in the venom gland of both species. Among the transcripts related with envenoming, high abundance of hyaluronidases was observed in both species. In addition, we built three-dimensional homology models based on several venom transcripts identified. Our study represents a significant improvement in the information about the venoms employed by these two species and their molecular characteristics. Moreover, the information generated by our group helps in a better understanding of the biology of freshwater cartilaginous fishes and offers clues for the development of clinical treatments for stingray envenoming in Brazil and around the world. Finally, our results might have biomedical implications in developing treatments for complex diseases.

Maturity-related changes in venom toxicity of the freshwater stingray Potamotrygon leopoldi.

Aquatic venomous animals such as stingrays represent a largely untapped source for venom-based drug development. However, the major challenge for a potential drug development pipeline is the high inter- and intraspecific variability in toxicity and venom composition. As of today, little is known about maturity-driven changes in these traits in stingrays. The present study investigates the differences in toxicity and venom composition in different maturity stages of the freshwater stingray Potamotrygon leopoldi. This species can be found in the Xingú River basin (Brazil), where it mainly feeds on invertebrates, while being predated by other stingrays or large catfishes. P. leopoldi, as commonly known for stingrays, can cause severe injuries with the venomous dentine spine located at its tails. The toxicity of tissue extracts of juvenile and mature specimens was recorded on a myoblast cell culture bioassay. Venom composition and bioactivity of compounds were analyzed with planar chromatography linked to an Aliivibrio fischeri bioassay. Results revealed a decrease in venom toxicity during maturation, but no changes in venom composition. These findings may indicate that toxicity in mature specimens becomes evolutionary less important, probably due to a decrease in predation pressure.

Systemic rhabdomyolysis induced by venom of freshwater stingrays Plesiotrygon iwamae and Potamotrygon motoro (Chondrichthyes-Potamotrygonidae) from the Amazon Basin.

Injuries caused by freshwater stingrays are characterized by intense pain and pathological changes at the lesion site, including oedema, erythema and, in most cases, necrosis. In this study, the systemic myotoxic activity induced by mucus extracts from the dorsal region and stinger of the stingrays Plesiotrygon iwamae and Potamotrygon motoro was described, analysed and quantified. Twenty-four hours after injection of 400 µg of the extracts into the gastrocnemius muscle of mice, the following effects were observed: coagulative necrosis of the muscle tissue, muscle fibre regeneration and the presence of inflammatory infiltrates, including neutrophils, macrophages, and a reduced number of eosinophils and lymphocytes. These changes were also observed, although to a lesser extent, in the gastrocnemius muscles of the contralateral limbs, demonstrating that the extracts from the two species could induce systemic rhabdomyolysis. Based on morphometric analysis, it was observed that the stinger extract of P. motoro was more potent in inducing local and systemic myotoxic activity, followed by the dorsal extract from P. motoro and stinger and dorsal extracts from P. iwamae, which induced similar effects.

Mercury in muscle and liver of ten ray species from Northwest Mexico.

We determined total mercury (Hg) distribution in muscle and liver of ten ray species and assessed health risk considering Hg levels in muscle and average consumption of rays in Mexico. Rays were collected from five sites in NW Mexico between March and November 2012. Hg concentrations ranged from 4.465 µg g(-1) in muscle of the longtail stingray Dasyatis longa to 0.036 µg g(-1) in liver of the diamond stingray Dasyatis dipterura. Considering all the individuals, Hg in muscle (1.612±1.322 µg g(-1)) was significantly (p<0.05) higher than in liver (0.745±0.616 µg g(-1)). Regarding local health risk assessment, none of the ray species may cause adverse effects on consumers.

Some euryhalinity may be more common than expected in marine elasmobranchs: the example of the South American skate Zapteryx brevirostris (Elasmobranchii, Rajiformes, Rhinobatidae).

Elasmobranchs are essentially marine, but ~15% of the species occur in brackish or freshwater. The Brazilian marine coastal skate Zapteryx brevirostris, non-reported in nearby estuaries, was submitted to 35, 25, 15, and 5 psu, for 6 or 12h (n=6). Plasma was assayed for osmolality, urea, and ions (Na(+), Cl(-), K(+), Mg(2+)). Muscle water content was determined, and the rectal gland, kidney and gills were removed for carbonic anhydrase (CA) and Na(+),K(+)-ATPase (NKA) activities. The skate survived to all treatments. Plasma osmolality and urea levels decreased respectively by 27% and 38% after 12h in 5 psu (with respect to levels when in seawater), but plasma Na(+), Cl(-), and Mg(2+) were well regulated. Plasma K(+) showed some conformation after 12h. Muscle hydration was maintained. Branchial CA and NKA did not respond to salinity. Rectal gland NKA decreased upon seawater dilution, while renal NKA increased. This skate was shown to be partially euryhaline. The analysis of plasma urea of elasmobranchs in brackish and freshwater versus salinity and time-allied to the widespread occurrence of some euryhalinity in the group-led us to revisit the hypothesis of a brackish water habitat for elasmobranch ancestors.

Potamotrygon cf. henlei stingray mucus: biochemical features of a novel antimicrobial protein.

Antimicrobial molecules are important components of the innate immune system in vertebrates. They have been studied widely in several fishes, but little is known about these defence factors in stingrays, which are thought to have less sophisticated adaptive immune systems when compared to other teleosts. Stingrays from the specie Potamotrygon cf. henlei are distributed throughout the rivers of central-west Brazil, being the cause of numerous envenomations occurring in the dry seasons. In a previous study, we reported that the mucus of the stingray P. cf. henlei shows antimicrobial effects. Here, to analyze the antimicrobial compounds from the mucus of P. cf. henlei, we employed solid-phase extraction, chromatographic separation followed by ESI-MS, and Edman degradation. A protein similar to the ß-chain of hemoglobin was identified, isolated and partially sequenced by Edman degradation. This protein has a molecular weight of 16072.8 Da, and was shown to be active against bacteria (Micrococcus luteus and Escherichiacoli) and yeast (Candida tropicalis) without hemolytic activity. Effects of this new protein in the microcirculation environment were also evaluated. The results obtained provide fundamental information for future basic research, clinical diagnosis and development of new therapies to accident treatment. To the best of our knowledge, this is the first description of a bioactive polypeptide from the mucus of a stingray.

Thorough analysis of polyhalogenated compounds in ray liver samples off the coast of Rio de Janeiro, Brazil.

INTRODUCTION: Five liver samples of two different ray species (Gymnura altavela and Zapteryx brevirostris) off the coast of Rio de Janeiro, Brazil, were analyzed for their pollution with anthropogenic and naturally occurring organohalogen compounds. MATERIAL AND METHODS: The samples were extracted with accelerated solvent extraction, and after a clean-up procedure, organohalogen compounds were separated by a modified group separation on activated silica. Subsequent analyses were done by targeted and non-targeted gas chromatography-mass spectrometry in the electron capture negative ion mode. RESULTS AND DISCUSSION: "Classic" organohalogen compounds such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and technical 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) were detected and quantified. PCBs generally exceeded the parts per million level and represented up to 90% of the total contamination of the ray livers. High concentrations were also detected for p,p'-DDE. Non-targeted full scan investigations lead to the detection of an abundant trichlorinated compound which was identified as a new DDT metabolite in biota. Different PBDE congeners and several halogenated natural products were quantified as well. In addition, polychlorinated terphenyls were identified and analyzed in the two species. Moreover, both ray species showed different fatty acid patterns and stable carbon isotope signatures. CONCLUSIONS: The two ray species showed high concentrations of organohalogen compounds in their liver tissue. Varied δ (13)C values by up to 3.1‰ indicated that the two ray species were living in different habitats.

The influence of environmental bacteria in freshwater stingray wound-healing.

Invasion by bacteria can influence the course of healing of wounds acquired in aquatic environment. In this study, the bacteria present in Potamotrygon motoro stingray mucus and in the Alto Paraná river water were identified, and their ability to induce tissue injury and resist antibiotics was determined. Biochemical identification analysis showed that 97% of all bacterial isolates were Gram negative, Aeromonas spp., Enterobacter cloacae and Citrobacter freundii being the species most prevalent. Gelatinase and caseinase were produced by Aeromonas hydrophila, Aeromonas sobria and Pseudomonas aeruginosa strains. Erythrocyte hemolysis assay showed that A. sobria, A. hydrophila and to a lesser extent, other Gram-negative bacteria produced hemolysin. It was also observed that molecules released in culture by these bacteria were toxic to human epithelial cells. Antibiogram results showed that 68% of all bacterial isolates were resistant to at least one type of antibiotic, mainly B-lactams. Finally, it was demonstrated that although P. motoro venom was toxic to epithelial cells it did not influence bacterial proliferation. In summary, the results obtained in this work indicate that during the accident, the mucus of P. motoro and the environmental water may transfer into the wound pathogenic multi-resistant bacteria with the potential to cause severe secondary infections.

Characterization of a new bioactive peptide from Potamotrygon gr. orbignyi freshwater stingray venom.

Brazilian freshwater stingrays, Potamotrygon gr. orbigyni, are relatively common in the middle-western regions of Brazil, where they are considered an important public health threat. In order to identify some of their naturally occurring toxin peptides available in very low amounts, we combine analytical protocols such as reversed-phase high-performance liquid chromatography (RP-HPLC), followed by a biological microcirculatory screening and mass spectrometry analysis. Using this approach, one bioactive peptide was identified and characterized, and two analogues were synthesized. The natural peptide named Porflan has the primary structure ESIVRPPPVEAKVEETPE (MW 2006.09 Da) and has no similarity with any bioactive peptide or protein found in public data banks. Bioassay protocols characterized peptides as presenting potent activity in a microcirculatory environment. The primary sequences and bioassay results, including interactions with the membrane phospholipids, suggest that these toxins are a new class of fish toxins, directly involved in the inflammatory processes of a stingray sting.

Mercury and arsenic in muscle and liver of the golden cownose ray, Rhinoptera steindachneri, Evermann and Jenkins, 1891, from the upper Gulf of California, México.

Mercury and arsenic levels in muscle and liver of Rhinoptera steindachneri were determined in organisms collected in 2006. Element concentrations in both tissues were directly related to size. Maxima mean concentrations of Hg and As (0.41 and 59.9 microg g(-1) dry wt, respectively) were found in adults muscle. Mercury concentrations were significantly different between juveniles and adults in muscle and liver. For As concentrations, differences between juveniles and adults were found only in muscle. Mercury concentrations were higher in muscle of juveniles and adults. Arsenic concentrations were higher in liver of juveniles, and in muscle of adults. Maximum Hg concentration in muscle (0.65 microg g(-1) dry wt) was below the safe limit established by Mexican regulations for seafood.

Ionic regulation and Na(+)-K(+)-ATPase activity in gills and kidney of the freshwater stingray Paratrygon aiereba living in white and blackwaters in the Amazon Basin.

During low-water period, freshwater stingray Paratrygon aiereba collected in the whitewater (WW) of the River Amazon showed higher urea content, osmolality, Na(+) and Cl(-) concentrations in plasma and perivisceral fluid than those caught in blackwater (BW) of the River Negro. Gills and kidney Na(+)-K(+)-ATPase activities were significantly lower in WW than in BW fish. The high level of kidney Na(+)-K(+)-ATPase activity in P. aiereba may minimize ion loss and generate diluted solute-free urine in ion-poor BW environment.

Nutritional value of various ray fish liver oils to the pacific white shrimp Litopenaeus vannamei.

A 32-day comparative feeding trial was performed to evaluate the nutritional value of four different ray fish liver oils to the Pacific white shrimp Litopenaeus vannamei. Four feeds were prepared with liver oil extracted from Dasyatis brevis, Rhinoptera steindachneri, Aetobatus narinari, and R. bonasus. A control feed was prepared with Menhaden fish oil. Ray fish liver oils were mainly composed of poly- and highly unsaturated fatty acids and contained levels of 20:5n-3 and 22:6n-3 comparable in magnitude to those of Menhaden fish oil, except for A. narinari liver oil, which had moderately low concentrations of 20:5n-3 and 22:6n-3 but showed a particularly high level of 20:4n-6, more than six times greater than that of Menhaden fish oil. Dietary fatty acids significantly influenced the fatty acid composition of shrimp muscle tissue, e.g., the diet with Menhaden fish oil elicited significantly higher shrimp muscle DHA level than diets with oil from D. brevis, and A. narinari, but not than diets with oil from R. steindachneri and R. bonasus. In spite of these differences, all four ray fish liver oils evaluated were as efficient in promoting growth and survival of L. vannamei as Menhaden fish oil, an ingredient known for its adequate nutritional quality to shrimp and fish. This study demonstrated one of the many possible applications of a locally-available resource that is currently being wasted.

Chalcedony (a crystalline variety of silica): biogenic origin in electric organs from living Psammobatis extenta (family Rajidae).

The electric organs of electric fish have been used extensively for the study of peripheral cholinergic synapses. Aluminum and silicon have been observed in the electrocytes of Psammobatis extenta, a fish belonging to the family Rajidae, using a combination of scanning electron microscopy and X-ray spectrometry. Based on this evidence, the presence of silica minerals has been documented by means of mineralogical techniques. Electric organ cryostat sections and subcellular fractions were observed using a Leica DMLP mineralogical microscope. The shape, size and color, among other properties, were analyzed in plane-polarized light, while birefringence and the extinction angle, which allow for mineral identification, were observed through crossed-polarized illumination. The distribution of chalcedony, an oxide silicon mineral, in the sections and all the fractions of the electric organ was recorded. X-ray diffraction analysis of the electric organ segments showed a similar result, with a low-quartz variety. Chalcedony precipitation occurred at a specific pH (7-8) and oxidation potential (Eh; 0.0 to -0.2). This observation supports the important role played by pH and Eh conditions in silica precipitation in electrocytes, as has been reported in geological environments. It is possible that silica formation and silica degradation in electric organs are also related to the enzymes, silicatein and silicase, that direct the polymerization and depolymerization of amorphous silica in sponges. Carbonic anhydrases (silicase) are involved in physiological pH regulation. Crystallization of chalcedony via spiral growth from a partially polymerized fluid is consistent with processes known to occur in organic systems. This is the first time that a biogenically produced crystalline mineral phase (i.e., chalcedony) has been observed in the electrocytes and cholinergic nerves from living electric fish.

Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays.

Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), tissue extracts have similar bands above 80 kDa, but most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive activity was verified in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue extract. No direct hemolysis, phospholipase A2 and coagulant activities were observed in both tissue extracts. Antigenic cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specific sera reacted with several components of both tissue extracts, noticeably above 22kDa. Both tissue extracts presented gelatinolytic, caseinolytic and fibrinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more severe in human accidents.