Morphology and phylogenetic relationships of a remarkable new genus and two new species of Neotropical freshwater stingrays from the Amazon basin (Chondrichthyes: Potamotrygonidae)

The morphology and phylogenetic relationships of a new genus and two new species of Neotropical freshwater stingrays, family Potamotrygonidae, are investigated and described in detail. The new genus, Heliotrygon, n. gen., and its two new species, Heliotrygon gomesi, n. sp. (type-species) and Heliotrygon rosai, n. sp., are compared to all genera and species of potamotrygonids, based on revisions in progress. Some of the derived features of Heliotrygon include its unique disc proportions (disc highly circular, convex anteriorly at snout region, its width and length very similar), extreme subdivision of suborbital canal (forming a complex honeycomb-like pattern anterolaterally on disc), stout and triangular pelvic girdle, extremely reduced caudal sting, basibranchial copula with very slender and acute anterior extension, and precerebral and frontoparietal fontanellae of about equal width, tapering very little posteriorly. Both new species can be distinguished by their unique color patterns: Heliotrygon gomesi is uniform gray to light tan or brownish dorsally, without distinct patterns, whereas Heliotrygon rosai is characterized by numerous white to creamy-white vermiculate markings over a light brown, tan or gray background color. Additional proportional characters that may further distinguish both species are also discussed. Morphological descriptions are provided for dermal denticles, ventral lateral-line canals, skeleton, and cranial, hyoid and mandibular muscles of Heliotrygon, which clearly corroborate it as the sister group of Paratrygon. Both genera share numerous derived features of the ventral lateral-line canals, neurocranium, scapulocoracoid, pectoral basals, clasper morphology, and specific patterns of the adductor mandibulae and spiracularis medialis muscles. Potamotrygon and Plesiotrygon are demonstrated to share derived characters of their ventral lateral-line canals, in addition to the presence of angular cartilages. Our morphological phylogeny is further corroborated by a molecular phylogenetic analysis of cytochrome b based on four sequences (637 base pairs in length), representing two distinct haplotypes for Heliotrygon gomesi. Parsimony analysis produced a single most parsimonious tree revealing Heliotrygon and Paratrygon as sister taxa (boot-strap proportion of 70%), which together are the sister group to a clade including Plesiotrygon and species of Potamotrygon. These unusual stingrays highlight that potamotrygonid diversity, both in terms of species composition and undetected morphological and molecular patterns, is still poorly known.

Diversification and Species Boundaries of Rhinebothrium (Cestoda; Rhinebothriidea) in South American Freshwater Stingrays (Batoidea; Potamotrygonidae)

Background: Neotropical freshwater stingrays (Batoidea: Potamotrygonidae) host a diverse parasite fauna, including cestodes. Both cestodes and their stingray hosts are marine-derived, but the taxonomy of this host/parasite system is poorly understood. Methodology: Morphological and molecular (Cytochrome oxidase I) data were used to investigate diversity in freshwater lineages of the cestode genus Rhinebothrium Linton, 1890. Results were based on a phylogenetic hypothesis for 74 COI sequences and morphological analysis of over 400 specimens. Cestodes studied were obtained from 888 individual potamotrygonids, representing 14 recognized and 18 potentially undescribed species from most river systems of South America. Results: Morphological species boundaries were based mainly on microthrix characters observed with scanning electron microscopy, and were supported by COI data. Four species were recognized, including two redescribed (Rhinebothrium copianullum and R. paratrygoni), and two newly described (R. brooksi n. sp. and R. fulbrighti n. sp.). Rhinebothrium paranaensis Menoret & Ivanov, 2009 is considered a junior synonym of R. paratrygoni because the morphological features of the two species overlap substantially. The diagnosis of Rhinebothrium Linton, 1890 is emended to accommodate the presence of marginal longitudinal septa observed in R. copianullum and R. brooksi n. sp. Patterns of host specificity and distribution ranged from use of few host species in few river basins, to use of as many as eight host species in multiple river basins. Significance: The level of intra-specific morphological variation observed in features such as total length and number of proglottids is unparalleled among other elasmobranch cestodes. This is attributed to the large representation of host and biogeographical samples. It is unclear whether the intra-specific morphological variation observed is unique to this freshwater system. Nonetheless, caution is urged when using morphological discontinuities to delimit elasmobranch cestode species because the amount of variation encountered is highly dependent on sample size and/or biogeographical representation.

Record of the freshwater stingrays Potamotrygon brachyura and P. motoro (Chondrichthyes, Potamotrygonidae) in the lower Uruguay river, South America

Freshwater stingrays, or potamotrygonids, are restricted to Neotropical river drainages. These elasmobranchs are well adapted to freshwater environments and the number of described species gradually increases as further research is carried out. Some of the first studies on their systematics and natural history were carried out in the 1960s and 1970s in southern South America. However, there is no new published data on potamotrygonids from Uruguayan waters since then (except for local journal reports from sportive fishermen and specimens deposited in Uruguayan collections). The present study aims to record the recent occurrence of two species of potamotrygonids caught by sport fishermen, with comments on other published historical records for the same area. As many other elasmobranchs, these species have an important, but not always well understood, role in the Uruguayan rivers ecosystems.

Preliminary data on the feeding habits of the freshwater stingrays Potamotrygon falkneri and Potamotrygon motoro (Potamotrygonidae) from the Upper Paraná River basin, Brazil

As raias da família Potamotrygonidae representam um grupo singular da ictiofauna Neotropical. Apesar de serem antigos os relatos sobre o grupo, ainda são muitas as questões que permanecem sem resposta, sobretudo no que diz respeito à biologia das espécies que ocorrem na Bacia do Paraná-Paraguai. No presente trabalho foi analisada a dieta de Potamotrygon falkneri e Potamotrygon motoro, capturadas no Alto Rio Paraná, a jusante da Usina Hidrelétrica Engenheiro Souza Dias (UHE Jupiá). As duas espécies de raias apresentaram dieta diversificada, ingerindo 14 itens, entre moluscos, crustáceos, insetos e peixes, porém com predominância de insetos aquáticos em diversidade e abundância. Somente um indivíduo de cada espécie ingeriu peixe. Potamotrygon motoro consumiu principalmente Ephemeroptera, enquanto P. falkneri, principalmente Mollusca, Hemiptera e Trichoptera. Os dados aparentemente indicam uma dieta mais especializada de P. motoro, com maior consumo de Ephemeroptera (Baetidae), e uma dieta mais generalizada de P. falkneri. A análise dos indivíduos capturados em três micro-hábitats, que diferem quanto ao tipo de substrato e presença de vegetação marginal, sugere diferenças nos tipos de alimentos consumidos.

An unusual, dwarf new species of Neotropical freshwater stingray, Plesiotrygon nana sp. nov., from the upper and mid Amazon basin: the second species of Plesiotrygon (Chondrichthyes: Potamotrygonidae)

A new species of the relatively poorly known Neotropical freshwater stingray genus Plesiotrygon Rosa, Castello & Thorson, 1987 is described from the main channel and smaller tributaries (Ríos Itaya and Pachitea) of the upper Amazon basin in Peru. The first specimen to be collected, however, was from much farther east in Rio Solimões in 1996, just down-river from Rio Purus (specimen unavailable for this study). Plesiotrygon nana sp. nov., is a very distinctive and unusually small species of freshwater stingray (Potamotrygonidae), described here mostly from three specimens representing different size classes and stages of sexual maturity. Plesiotrygon nana sp. nov., is distinguished from its only congener, P. iwamae Rosa, Castello & Thorson, 1987, by numerous unique features, including: dorsal coloration composed of very fine rosettes or a combination of spots and irregular ocelli; very circular disc and snout; very small and less rhomboidal spiracles; short snout and anterior disc region; narrow mouth and nostrils; denticles on dorsal tail small, scattered, not forming row of enlarged spines; adult and preadult specimens with significantly fewer tooth rows; fewer caudal vertebrae; higher total pectoral radials; very small size, probably not surpassing 250 mm disc length or width, males maturing sexually at around 180 mm disc length and 175 mm disc width; distal coloration of tail posterior to caudal stings usually dark purplish-brown; and features of the ventral lateral-line canals (hyomandibular canal very narrow, infraorbital and supraorbital canals not undulated, supraorbital and infraorbital loops small and narrow, supraorbital loop very short, not extending posteriorly to level of mouth, jugular and posterior infraorbital canals short, not extending caudally to first gill slits, subpleural loop very narrow posteriorly; absence of anterior and posterior subpleural tubules). To provide a foundation for the description of P. nana sp. nov., morphological variation in P. iwamae was examined based on all type specimens as well as newly collected and previously unreported material. Two specimens topotypic with the male paratype of P. nana sp. nov., referred to here as Plesiotrygon cf. iwamae, are also reported. Relationships of the new species to P. iwamae are discussed; further characters indicative of Plesiotrygon monophyly are proposed, but the genus may still not be valid. Plesiotrygon nana sp. nov., is commercialized with some regularity in the international aquarium trade from Iquitos (Peru), an alarming circumstance because nothing is known of its biology or conservation requirements.

A new species of Neotropical freshwater stingray of the genus Potamotrygon Garman, 1877 from the Río Madrede Díos, Peru (Chondrichthyes: Potamotrygonidae)

Potamotrygon tatianae sp. nov., is described from Río Madre de Díos, Peru, upper Rio Madeira basin. The new species is distinguished from all congeners by a unique combination of characters, including its dorsal color pattern formed by a relatively slender, highly convoluted, beige to dark brown vermicular pattern, a single row of dorsal tail spines, and a relatively longer tail posterior to caudal stings. Potamotrygon tatianae sp. nov., occurs sympatrically with other species of Potamotrygon (P. falkneri, P. orbignyi and P. motoro). From the similar species P. falkneri, P. tatianae sp. nov., is further distinguished by the absence of circular, reniform, and oval spots, by its proportionally much longer tail, by having dorsal tail spines in one irregular row, and by features of the ventral lateral-line canal, dermal denticles and neurocranium. From P. orbignyi, the new species is distinct by lacking a reticulate pattern on dorsal disc and by the presence of two angular cartilages. From P. motoro, P. tatianae sp. nov., is further separated by the lack of ocelli formed by strong black concentric rings, by the more flattened aspect of its head and disc, and by having smaller and more numerous teeth. The discovery of a new species that so closely resembles a congeneric form in color pattern, a feature highly variable within the latter, highlights the importance of examining large series of individuals and of detailed morphological analyses in revealing the potentially highly cryptic nature of the diversity within the family.

Potamotrygon boesemani (Chondrichthyes: Myliobatiformes: Potamotrygonidae), a new species of Neotropical freshwater stingray from Surinam

Potamotrygon boesemani, new species, is described from the Corantijn river drainage in Surinam. The species has a diagnostic dorsal color pattern formed by deep orange to red ocellated spots of irregular form, encircled by relatively broad black rings. Potamotrygon boesemani is distinguished from other ocellated congeners (P. motoro, P. henlei and P. leopoldi) by the more intensely colored ocelli, which are usually yellow in the latter species. From P. motoro it is also distinguished by the darker dorsal background coloration, by the broader black contour of the dorsal ocelli, and by the irregular form of the ocelli as compared to the more rounded shape in the latter species. From P. henlei and P. leopoldi, it is distinguished by the lack of ocelli on tail. From the tentatively identified specimen of P. ocellata, which also has dark orange ocelli, the irregular contour of the ocelli in the new species is also distinctive. The teeth are relatively smaller and in greater number than in P. motoro and P. ocellata, with up to 45 rows in the upper jaw.

Histopathological changes induced by extracts from the tissue covering the stingers of Potamotrygon falkneri freshwater stingrays

Pain is the most conspicuous symptom observed in patients wounded by stingrays, and skin necrosis is common in accidents by freshwater stingrays. The extract from the stinger integumentary tissue of Potamotrygon falkneri containing toxic components (venom) was tested for its ability to induce histopathological changes in the dorsal skin of mice at different times. 3-6 h after injection, foci of necrosis in isolated basal epidermal cells were observed. Full coagulative necrosis of the skin, subcutaneous tissue and skeletal muscle was evident as soon as 24 h after venom exposure, with a clear demarcation from the normal skin. After 48 h, round collections of necrotic cells start to coalesce originating extensive skin necrotic plaques that detach from viable tissue after 72-96 h. Inflammatory infiltrate was observed after 6 h, but was always mild. Acute vascular thrombosis was rare, and hemorrhage was not present at any time. Superficial bacterial infection was present in two of the examined cases. In conclusion, the venom of P. falkneri is responsible for the development of an early necrosis with mild inflammatory reaction, probably due to direct action of the venom. The severe local damage is probably worsened by the mechanical trauma caused by the stinger. (c) 2010 Elsevier Ltd. All rights reserved.

Scolex Morphology of Proteocephalid Cestodes Parasites of Neotropical Freshwater Fishes

The morphology of the scolices and metascolices of 29 proteocephalid species, parasites of freshwater fishes from Brazil was compared by using scanning electron microscope, light and laser microscopy. The following species were evaluated: Proteocephalus vazzolerae, P. piramutab, Robertiella paranaensis, Travassiella avitellina, Monticellia loyolai, M. spinulifera, M. belavistensis, Houssayela sudobim, Zygobothrium megacephalum, Gibsoniela mandube, Cangatiella arandasi, Nomimoscolex sudobim, N. lopesi, N. admonticellia, N. piraeeba, N. pirarara, Harriscolex kaparari, Crepidobothrium eirasi, Spatulifer rugosa, Megathylacus brooksi, Choanoscolex abscisus, Amphoteromorphus peniculus, A. piraeeba, Paramonticellia itaipuensis, Peltidocotyle rugosa, Othinoscolex lenha, Rudolphiella rugata, R. piranabu, Jauella glandicephalus. Some features as overall shape of scolex, suckers, apical sucker, frontal glands and several kinds of metascolex were analyzed. The taxonomic importance of scolex and metascolex is discussed.

Acute lethal and sublethal effects of neem leaf extract on the neotropical freshwater fish Prochilodus lineatus

The aim of this study was to determine the toxicity of the aqueous extract of neem leaves, a product extensively used in fish-farms as alternative for the control of fish parasites and fish fry predators, for the neotropical fish Prochilodus lineatus. The 24 It LC(50) of neem leaf extract for juveniles P lineatus was estimated as 4.8 g L(-1); the fish were then exposed for 24 h to 2.5, 5.0 and 7.5 g L(-1) or only clean water (control). Plasma glucose levels were higher in fish exposed to 2.5 g L(-1) and 5.0 g L(-1) neem extract, relative to control, indicating a typical stress response. Neem extract did not interfere with the osmoregulating capacity of the fish, as their plasma sodium, chloride, total protein and osmolarity did not change. The presence of the biopesticide interfered with the antioxidant defense system of P. lineatus, as there was a decrease in liver catalase activity at all neem concentrations and the detoxifying enzyme glutathione-S-transferase was activated in fish exposed to 5.0 g L(-1). Fish exposed to all neem extract concentrations exhibited damaged gill and kidney tissue. These results indicate that although neem extract is less toxic to P. lineatus than other synthetic insecticides used in fish-farming it does cause functional and morphological changes in this fish species. (c) 2006 Elsevier B.V. All rights reserved.

Hematological parameters in two neotropical freshwater teleost, Leporinus macrocephalus (Anostomidae) and Prochilodus lineatus (Prochilodontidae)

Leporinus macrocephalus is a non-migratory omnivore fish which occurs in waters with a relatively high oxygen rate. Prochilodus lineatus is a migratory fish that inhabits deoxygenated hypolimnion and feeds on detritus material. Red blood cell, thrombocytes and white blood cell counts, hematocrit and hemoglobin, serum electrolytes (sodium, potassium, calcium, magnesium and chloride), metabolic products (total serum protein and plasma glucose) and blood cells major axis length for L. macrocephalus and P. lineatus were compared. White blood cell counts for both species indicated a similar activity of the immune functions. The concentration of total protein, sodium, calcium, magnesium and chloride, MCV, as well as red blood cells, monocytes, and thrombocytes size in L. macrocephalus were higher than in P. lineatus. However, P. lineatus had higher red blood cell counts, hematocrit and hemoglobin, which reflects a considerable adaptation to survive in an environment with low levels of oxygen.

Freshwater stingrays: study of epidemiologic, clinic and therapeutic aspects based on 84 envenomings in humans and some enzymatic activities of the venom

Freshwater stingrays are very common in the Parana, Paraguay, Araguaia, and Tocantins Rivers and tributaries in Brazil. This study presents the clinical aspects of 84 patients injured by freshwater stingrays. Intense pain was the most conspicuous symptom. Skin necrosis was observed in a high percentage of the victims, mostly fishermen and bathers. The initial therapeutic procedures, like immersion of the affected member in hot water were effective in the initial phases of the envenoming, especially in the control of the acute pain; however, they did not prevent skin necrosis. By SDS-PAGE, the freshwater stingray (Potamotrygon falkneri) venom extract presented a major band of approximately 12 kDa. Several other components distributed between 15 and 130 kDa were detected in the venom extract. Many components with molecular mass above 80 and 100 kDa have gelatinolytic and caseinolytic activities, respectively. Hyaluronidase activity was detected only in a component around 84 kDa in P. falkneri venom extract. Our results demonstrated that the presence of these enzymes could explain partially the local clinical pictures presented by patients wounded by freshwater stingray. (C) 2004 Elsevier Ltd. All rights reserved.

DNA barcoding reveals hidden diversity in the Neotropical freshwater fish Piabina argentea (Characiformes: Characidae) from the Upper Parana Basin of Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The 5S rDNA family evolves through concerted and birth-and-death evolution in fish genomes: an example from freshwater stingrays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)