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We tracked thirteen giant otters from three different groups in the Balbina Hydroelectric Reservoir. One of them was a transient animal, while the others were living in groups of five and seven individuals. Travelling was the most frequent behaviour, both for the transient otter (>40%) and group otters (46%). Diurnal resting was the longest-lasting behaviour. Most giant otter behaviour in the reservoir showed little difference when compared with the behaviour of giant otters living in nondammed areas, indicating a significant degree of resilience of this species. However, otters in the reservoir spent more time travelling, probably because their territory sizes were much greater than the territories of giant otters in nondammed areas. The frequency of fishing was similar to what has been recorded elsewhere, suggesting that current fish density and availability in the reservoir is similar to what is observed in nondammed areas. Nevertheless, otters in Balbina spent more time fishing than those in nondammed areas, which alternatively could suggest that fish density in the reservoir was low and could be compensated by increasing the time spent fishing. The high frequency of intraspecific kleptoparasitism observed in this study (62%) is noteworthy, although virtually undocumented for this species. This is the first study of giant otter behaviour in a hydroelectric reservoir that can be used for the conservation of this endangered species in man-made lakes.

Studies on the microbiota of freshwater cetaceans are scarce and may provide important data on animal and environmental health. This study aimed to evaluate the antimicrobial susceptibility of Gram-negative bacteria recovered from two populations of free-ranging Amazon river dolphins (Inia geoffrensis). Twenty-one animals were captured and released, 13 from Negro River and 8 from Tapajos River, Brazil. Swab samples were obtained from the oral cavity, blowhole, genital opening and rectum and were cultured on MacConkey agar. Isolates were biochemically identified, and antimicrobial susceptibility was assessed by disk diffusion method. Overall, 132 isolates were recovered, of which 71 were recovered from animals from Negro River and 61 from Tapajos River. The most commonly recovered bacterial species were Enterobacter cloacae, Morganella morganii, Klebsiella pneumoniae and Pseudomonas aeruginosa. Overall, 51.6% (63/122) of the isolates were not-susceptible (intermediate resistance and resistance), of which 28/122 (22.9%) were resistant to at least one antimicrobial. Cephalothin, cefuroxime and cefepime were the drugs to which more resistant and intermediate results were observed (P

Body length is an important parameter in morphological, ecological and behavioral studies of a species and contributes to the understanding of the body condition of individuals. This parameter is essential for conservation and management strategies by informing studies evaluating growth rates, physical maturity and classification of individuals into age groups, promoting better accuracy for the biological parameters of the species. The aim of this study was to identify predictors of body length for Amazonian manatees (Trichechus inunguis) using metric characters of osteological materials. Eleven linear measurements of skull, jaw, scapulae and humeri were collected from 41 Amazonian manatee skeletons of different age classes (calf, juvenile and adult). Data were analyzed by simple linear regression. The condylobasal length was the best predictor of body length for the species (R = 0.943), however, all bones evaluated showed at least one measurement with the capacity to predict body size (R2 > 0.9). Results of this study are useful for inferring body length of Amazonian manatees using bones deposited in museums and biological collections, expanding the informative potential of these materials. RESUMO O comprimento corporal é um importante parâmetro em estudos morfológicos, ecológicos e comportamentais de uma espécie e contribui para o entendimento da condição corporal de um indivíduo. Este parâmetro é essencial para estratégias de conservação e manejo, informando estudos que avaliam taxas de crescimento, maturidade física e classificação dos indivíduos em grupos de idade, promovendo uma melhor precisão aos parâmetros biológicos de uma espécie. O objetivo desse trabalho foi identificar preditores de comprimento corporal para o peixe-boi da Amazônia (Trichechus inunguis) utilizando caracteres métricos de material osteológico. Onze medidas lineares do crânio, mandíbula, escápula e úmero foram coletadas de 41 esqueletos de peixe-boi da Amazônia de diferentes classes de idade (filhote, juvenil e adulto). Os dados foram analisados por regressão linear simples. O comprimento côndilobasal foi o melhor preditor de comprimento corporal para a espécie (R = 0,943), entretanto, todos os ossos avaliados mostraram pelo menos um caractere com capacidade de predizer o comprimento corporal (R2 > 0,9). Os resultados deste estudo são úteis para inferir o comprimento corporal de peixes-boi da Amazônia a partir de ossos depositados em museus e coleções biológicas, expandindo o potencial informativo destes materiais.

P-glycoprotein (P-gp) induction and/or activation have been proposed as therapeutic strategies in intoxication scenarios, by reducing the intestinal absorption of xenobiotics, including drugs. Oxygenated xanthones (OXs) have been described as P-gp modulators and, therefore, the main goals of this study were to: (a) investigate the potential modulatory effect of six OXs on P-gp expression and activity in SW480 cells; (b) validate these cells for the screening/identification of P-gp inducers/activators; (c) explore the potential OXs-mediated protective effects against the cytotoxicity of mitoxantrone (MTX), a toxic P-gp substrate. Four OXs (OX2, OX4, OX5, and OX6) increased P-gp expression 24 h after exposure. However, a lack of correlation between P-gp expression and activity was observed for OX1 and OX4. In addition, after a short incubation with the P-gp fluorescent substrate, rhodamine 123, all the studied OXs, except OX3, efficient and immediately increased P-gp activity, suggesting their potential as P-gp activators. Despite these results, OXs failed to afford protection against MTX-induced cytotoxicity. Docking simulations performed in a human P-gp model revealed that the lack of protection may be explained by the different binding locations of OXs and MTX within the P-gp drug-binding pocket. In conclusion, the in vitro results confirmed OXs potential for P-gp induction and/or activation and suggested SW480 cells as a suitable in vitro model for these studies. However, P-gp activation did not protected SW480 cells against MTX cytotoxicity. In silico studies suggested the different binding locations as a limiting step in the P-gp-mediated efflux of its substrates under P-gp activation.

We assessed the feeding habits of the Amazonian manatee inhabiting blackwater (Negro River) and clearwater (Tapajos River) igapos (floodplains) using δ13C and δ15N of dentin and bone collagen from different ontogenetic classes (nursing calves, juveniles, and adults). Within an individual, the dentine δ13C and δ15N values did not vary with tooth position. Bones were more depleted in 13C and 15N compared to teeth, and the δ13C and δ15N in bone differed among classes. Food sources had δ13C values typical of algae, plants of C3 or C4 photosynthetic pathways. Mixing models showed that lactating females (inferred by isotopic values from nursing calves) had higher proportional consumption of C4 plants, while, for other adults, C3 plants were more frequent in the diet of manatees from both rivers. Juveniles had a mixed diet of C3 pants and C4 plants. We hypothesize the C4 plants signal of calves results from female movements to the floodplains of nearby whitewater rivers (varzeas), where C4 plants are abundant. Individuals from Tapajos may be more resident, as C3 plants and C4 plants are available during the flood pulse. Results demonstrate that manatee diets vary with ontogenetic classes and water typology. Preservation of habitats (igapo/varzea) is therefore required for the survival of Amazonian manatees.

Herpesvirus (HV) infections in cetaceans are frequently associated with skin and mucosal lesions. Although HV infections have been reported worldwide, their occurrence in southern Atlantic marine mammals is still poorly understood. We tested skin, oral and genital mucosal beta-actin PCR-positive samples from 109 free-ranging Brazilian cetaceans using a universal herpesvirus DNA polymerase PCR. Herpesvirus-positive skin samples from a Guiana dolphin (Sotalia guianensis), a dwarf sperm whale (Kogia sima), a Bolivian river dolphin (Inia boliviensis), and a lingual sample from an Atlantic spotted dolphin (Stenella frontalis) were histologically evaluated. Additional tissue samples from these animals were also PCR-positive for HV, including a novel sequence obtained from the dwarf sperm whale's stomach and mesenteric lymph node. Four novel HV species were detected in the Guiana dolphin (one), the dwarf sperm whale (two) and the Bolivian river dolphin (one). The cutaneous lesions (marked, focally extensive, chronic proliferative dermatitis) of the Guiana dolphin and the Bolivian river dolphin were similar to previous HV reports in cetaceans, despite the absence of intranuclear inclusion bodies. This is the largest HV survey in South American cetaceans and the first detection of HV infection in riverine dolphins worldwide.

Introduction: P-glycoprotein (P-gp) is an important efflux pump responsible for the extruding of many endogenous and exogenous substances out of the cells. P-gp can be modulated by different molecules – including xanthone derivatives – to surpass the multidrug resistance (MDR) phenomenon through P-gp inhibition, or to serve as an antidotal strategy in intoxication scenarios through P-gp induction/activation. Areas covered: This review provides a perspective on P-gp modulators, with particular focus on xanthonic derivatives, highlighting their ability to modulate P-gp expression and/or activity, and the potential impact of these effects on the pharmacokinetics, pharmacodynamics and toxicity of P-gp substrates. Expert opinion: Xanthones, of natural or synthetic origin, are able to modulate P-gp, interfering with its protein synthesis or with its mechanism of action, by decreasing or increasing its efflux capacity. These modulatory effects make the xanthonic scaffold a promising source of new derivatives with therapeutic potential. However, the mechanisms beyond the xanthones-mediated P-gp modulation and the chemical characteristics that make them more potent P-gp inhibitors or inducers/activators are still understudied. Furthermore, a new window of opportunity exists in the neuropathologies field, where xanthonic derivatives with potential to modulate P-gp should be further explored to optimize the prevention/treatment of brain pathologies.

The two Sotalia species (the marine S. guianensis and the freshwater S. fluviatilis) have only recently been recognized, and both face several conservation challenges. We investigated the existence of hybridization between the two species in their possible area of sympatry in the Amazon Estuary, in northern Brazil. A fast and cheap PCR-RFLP diagnostic method using nuclear DNA was developed to discriminate between the two species, while allowing the detection of hybrids. All samples that could be identified (N = 51) were identified as S. guianensis, and no hybrids were detected. Our results, coupled with previous mitochondrial data, suggest that S. fluviatilis is not present in the Amazon delta. Thus, sympatry with S. guianensis, if it does occur, may be restricted to upstream areas of the Amazon River.

This work relied on the use microcosms to evaluate the individual and the combined effects of different levels of copper sulfate (0.0, 0.013, 0.064 and 0.318 mg Cu L−1) – a fungicide commonly exceeding allowable thresholds in agricultural areas – and a range of water temperature increase scenarios (15, 20 and 25 °C) on freshwater species belonging to different functional groups. Hence, the growth inhibition of primary producers (the microalgae Raphidocelis subcapitata and the macrophyte Lemna minor), as well as the survival and feeding behavior of a shredder species (the Trichoptera Schizopelex sp.) were evaluated. The results revealed that copper was toxic to primary producers growth, as well as shredders growth and survival, being the growth of L. minor particularly affected. Higher water temperatures had generally enhanced the growth of primary producers under non-contaminated (microalgae and macrophytes) or low-contaminated (macrophytes) conditions. Despite the tendency for a more pronounced toxicity of copper under increasing water temperatures, a significant interaction between the two factors was only observed for microalgae. Since the test organisms represent relevant functional groups for sustaining freshwater systems functions, the present results may raise some concerns on the impacts caused by possible future climate change scenarios in aquatic habitats chronically exposed to the frequent or intensive use of the fungicide copper sulfate.

The spermatozoa from seven adult Amazon river dolphins (Inia geoffrensis, CETACEA: INIIDAE) were analyzed by light and electron microscopy. The spermatozoa showed an elongated ellipsoid shaped head and a long tail with a well distinguishable midpiece. The head spermatozoa have a smooth surface like other odontocetes examined, with the exception of the Delphinidae family. The mean dimensions of the spermatozoa were within the range already reported for other cetaceans. The spermatozoa midpiece, as in other cetaceans, showed a random pattern of mitochondria, different from that described for other mammals. Further studies of sperm morphology of a wider spectrum of cetacean families could help to better understand the reproductive biology of these animals and the intergeneric and intrageneric relationships among them, as well as, among other mammals. Anat Rec, 300:1519-1523, 2017. © 2017 Wiley Periodicals, Inc.

The number of wildfires has markedly increased in Mediterranean Europe, including in Portugal. Wildfires are environmentally concerning, not only due to the loss of biodiversity and forest area, but also as a consequence of environmental contamination by specific compounds including metals and polycyclic aromatic compounds (PAHs). These contaminants, mostly bound to ashes, can reach downstream water bodies, namely through surface runoff, being ultimately dispersed by vast areas and contacting with aquatic biota. Being toxicologically noteworthy, the potential toxic outcomes of the input of such chemicals across the aquatic compartment must be characterized. In this context, the present study used a biomarker-based approach to find early-warning signals of toxicity triggered by the exposure of the mosquitofish, Gambusia holbrooki, to affected aqueous runoff and stream water samples collected from a forest burnt area. The chemical analysis revealed concerning levels of metals and polycyclic aromatic hydrocarbons in both runoff and stream water samples. Biological responses elicited by the collected samples showed the occurrence of pro-oxidative modifications, specifically driven by enzymatic forms involved in the metabolism of glutathione. Despite these effects, no further signs of involvement of metals and PAHs were elicited in terms of neurotoxicity. The overall set of data implicates chemicals resulting from wildfires in clear deleterious effects in exposed fish. published

P-glycoprotein (P-gp) plays a crucial role in the protection of susceptible organs, by significantly decreasing the absorption/distribution of harmful xenobiotics and, consequently, their toxicity. Therefore, P-gp has been proposed as a potential antidotal pathway, when activated and/or induced. Knowing that xanthones are known to interact with P-gp, the main goal was to study P-gp induction or/and activation by six new oxygenated xanthones (OX 1-6). Furthermore, the potential protection of Caco-2 cells against paraquat cytotoxicity was also assessed. The most promising compound was further tested for its ability to increase P-gp activity ex vivo, using everted intestinal sacs from adult Wistar-Han rats. The oxygenated xanthones interacted with P-gp in vitro, increasing P-gp expression and/or activity 24 h after exposure. Additionally, after a short-incubation period, several xanthones were identified as P-gp activators, as they immediately increased P-gp activity. Moreover, some xanthones decreased PQ cytotoxicity towards Caco-2 cells, an effect prevented under P-gp inhibition. Ex vivo, a significant increase in P-gp activity was observed in the presence of OX6, which was selectively blocked by a model P-gp inhibitor, zosuquidar, confirming the in vitro results. Docking simulations between a validated P-gp model and the tested xanthones predicted these interactions, and these compounds also fitted onto previously described P-gp induction and activation pharmacophores. In conclusion, the in vitro, ex vivo, and in silico results suggest the potential of some of the oxygenated xanthones in the modulation of P-gp, disclosing new perspectives in the therapeutics of intoxications by P-gp substrates.

The potential value of protected areas for the conservation of cetaceans is widely recognized; however, few evaluation methods exist to assess their effectiveness. In this study, a modeling approach based on long-term mark-recapture/resight data was used to assess the effectiveness of a Brazilian reserve in protecting endangered Amazon River dolphins or boto (Inia geoffrensis), a species killed for use as fish bait. We built an annual discrete-time model with subdivisions based on hydrological periods and age classes. It included transition probabilities in and out of the reserve that were estimated utilizing multi-state mark-recapture models. To evaluate five reserve configurations, we re-estimated the transition probabilities to represent changes in the reserve boundaries. Model predictions showed that four scenarios, including one representing the existing boundaries, would be insufficient to protect the local boto population (no = 528) and a steep decline in abundance would occur in the next 50 years (0 ≤ n50 ≤ 108). However, one reserve configuration, encompassing both flooded forests and adjacent river habitats, and including beaches and channel/floodplain entrances, resulted in a nearly stable population in the same time frame (n50 = 515). These findings suggest that, with careful design and efficient management, protected areas could be an effective conservation tool for boto populations that exhibit site fidelity to Amazonian floodplains. With appropriate mark-recapture/resight data, the modeling framework employed could be extended to evaluate and design protected areas for populations of other species in other systems.

Wildfires, increasingly frequent and severe in the Mediterranean countries, can cause serious impacts on ecosystems. As a diffuse source of contaminants – namely inorganic elements (metals, metalloids and non-metallic elements) and polycyclic aromatic hydrocarbons (PAHs) – post-fire runoff may cause toxic effects on the aquatic biota. Despite the recognition of these likely impacts, this issue has been still largely neglected. This study explores the impacts of ash from wildfires on the freshwater clam Corbicula fluminea and the ability of this filter feeder to concentrate the contaminants associated with ash in post-fire runoff. The bio-concentration potential of the clam was assessed in a 7-day filtration experiment, using aqueous extract of ashes (AEA) to represent post-fire runoff. Despite the noticeable mortality of the clams exposed to the AEA (∼30% higher in AEA than in control after 7 days of exposure), there was a clear reduction on the concentration of almost all pyrolytic substances in the water linked to bio-filtration and bio-concentration activity. Along 7 days the clams operated decreases of more than 70% for some chemicals (Cu, Mn and Zn) while others were no longer detected in the water at the end of the experiment (Cr, NAP and PHE). The removal from water was accompanied by contaminants’ accumulation in clams’ soft tissues and, less extensively, in shells. Lower pollutant loads of filtered AEA translated into decreased toxicity of the matrix towards standard aquatic species (microalgae, macrophytes and cladocerans) confirming an improvement as well in the biological quality of the water through bio-filtration. Although post-fire loads can compromise C. fluminea survival, this study evidenced the potential of this invasive species to contribute to water quality improvement.

Deliberate killing for use as bait in a regional catfish (Calophysus macropterus) fishery is the primary threat affecting the survival of the Amazon river dolphin, or boto (Inia geoffrensis). Establishing and improving freshwater protected areas has been suggested as a possible course of action to protect the species. However, the ecology of the boto is poorly understood and more information is needed on the species’ habitat use and movement patterns to ensure that spatial protection initiatives meet conservation needs. In this study, mark-recapture/resight data collected in and near the Mamiraua Sustainable Development Reserve, a varzea in the Brazilian Amazon, was used to examine transition probabilities of botos between various habitat types. Our findings suggest that it is imperative that spatial protection initiatives focused on the boto take into account the importance of varzea habitat and bay systems for individuals in early life stages. Moreover, because botos frequently occupy the main rivers, particularly during the low water period, protecting areas of the main river waters adjacent to varzeas could aid in the protection of hunted populations.

Wildfires can cause immediate and drastic impacts on the structure and functioning of ecosystems, and there has been an increasing interest in wildfire effects on water chemistry and aquatic biota. Wildfires are increasingly recognized as a diffuse source of contamination of aquatic ecosystems, through the production of deleterious pyrolytic substances and their subsequent transport, mostly attached to ashes. To study the deleterious effects of the ash-laden runoff from burnt areas on water quality, composite ash samples of ashes were collected immediately after a forest fire and then used to prepare aqueous extracts of ash (AEA). The AEA were analyzed with respect to a large group of chemical elements and the sixteen prioritized polycyclic aromatic hydrocarbons (PAHs). Ca, S, Mg, K and Na were found to be the principal elements in the AEA, while only two low molecular weight PAHs (phenanthrene and naphthalene) were present in quantifiable amounts. In parallel, an ecotoxicological screening of the AEA was performed with four standard aquatic species from different functional groups and trophic levels. The AEA was found to induce a statistically significant decrease in the growth of two primary producers – Pseudokirchneriella subcapitata and Lemna minor – and inhibited the luminescence of the bacteria Vibrio fischeri . By contrast, AEA did not produce a significant immobilization of Daphnia magna suggesting that short-term acute toxicity may be absent at higher trophic levels. Overall, the present results emphasize the role of wildfires as a potential source of diffuse contamination for downstream water bodies, compromising both chemical and ecological conditions. At the same time, this study highlights the need for further research into the complexity of the potentially deleterious ecological effects of wildfires on aquatic communities, with a particular focus on cascading effects along the trophic web.

ABC (ATP-binding cassette) transporters represent one of the largest and most diverse superfamily of proteins in living species, playing an important role in many biological processes such as cell homeostasis, cell signaling, drug metabolism and nutrient uptake. Moreover, using the energy generated from ATP hydrolysis, they mediate the efflux of endogenous and exogenous substrates from inside the cells, thereby reducing their intracellular accumulation. At present, 48 ABC transporters have been identified in humans, which were classified into 7 different subfamilies (A to G) according to their phylogenetic analysis. Nevertheless, the most studied members with importance in drug therapeutic efficacy and toxicity include P-glycoprotein (P-gp), a member of the ABCB subfamily, the multidrug-associated proteins (MPRs), members of the ABCC subfamily, and breast cancer resistance protein (BCRP), a member of the ABCG subfamily. They exhibit ubiquitous expression throughout the human body, with a special relevance in barrier tissues like the blood-brain barrier (BBB). At this level, they play a physiological function in tissue protection by reducing or limiting the brain accumulation of neurotoxins. Furthermore, dysfunction of ABC transporters, at expression and/or activity level, has been associated with many neurological diseases, including epilepsy, multiple sclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis. Additionally, these transporters are strikingly associated with the pharmacoresistance to central nervous system (CNS) acting drugs, because they contribute to the decrease in drug bioavailability. This article reviews the signaling pathways that regulate the expression and activity of P-gp, BCRP and MRPs subfamilies of transporters, with particular attention at the BBB level, and their mis-regulation in neurological disorders.

An adult male Amazonian manatee Trichechus inunguis under human care presented with 3 circular cutaneous lesions on the dorsal aspect of the rostrum and between the nostrils (plenum). Initially these lesions were superficial, hypopigmented, without warmth and non-painful. Microbiological cultures of skin swabs isolated Candida sp. and Pseudomonas aeruginosa, and topical treatment with antiseptic, antifungal, anti-inflammatory and antibiotic medication was instituted. This treatment strategy did not lead to any clinical improvement, and after 6 mo, the lesions progressed to a confluent abscess (5.0 × 3.0 cm) with increased temperature and obvious discomfort on palpation. An impression smear of a cutaneous biopsy was submitted for Ziehl-Neelsen staining and after detection of acid-fast bacilli, the cutaneous biopsy and a swab from the lesion were sent for histopathology, culture and sensitivity testing. After 5 d of incubation and through PCR-restriction analysis of the isolates, Mycobacterium fortuitum and M. abscessus were identified. Sensitivity testing indicated that the isolates were susceptible to ciprofloxacin and clarithromycin, and after draining of the lesion and administration of systemic antibiotic treatment, there was rapid clinical improvement. This report describes non-healing lesions in an aquatic animal and illustrates the importance of evaluating the presence of non-tuberculous mycobacteria, opportunistic pathogens which are ubiquitous in the aquatic environment, in protracted, non-responsive cases. We also highlight the importance of a correct diagnosis and treatment approach, and we review concerns that these bacteria are zoonotic agents and are frequently resistant to conventional antibiotics. © Inter-Research 2018.

We monitored the underwater behavior of botos (Inia geoffrensis) using stereo acoustic data loggers to observe their local habitat use and its diel changes at the Mamiraua Sustainable Development Reserve, Brazil. A-tags were set at five sites in three different habitat types: Lake (low current), Channel (middle current), and Junction (junction of two channels). The presence index during nighttime was significantly greater than during daytime in the Lake and Junction. Underwater movement was estimated from the changing pattern (trajectory) of the relative angle of the sound source from A-tags. A staying-type trajectory was dominant in the Lake, although the prevalence of moving-type trajectory increased at night. More than 80% of detected trajectories were the staying type in the Junction, while moving-type trajectories dominated in the Channel. The frequency of click trains was greatest in the Lake, followed by the Junction and Channels. The average interpulse interval, which reflects the mean target distance of echolocation, was shortest in the Lake, followed by the Junction and Channel. These results suggest that the botos used the Lake as their primary habitat for active behaviors like foraging, especially at night, and the Junction as their primary habitat for relatively inactive behaviors at night.

Teeth and otoliths are metabolically inert structures that preserve a chronology of chemical variations that may be related to the environmental histories experienced by each organism. Because of the natural decrease of barium (Ba) and increase of strontium (Sr) bioavail- ability in water with increasing salinity, these elements may be especially useful to track habitat use in aquatic organisms. Therefore, we tested whether the Ba/Ca and Sr/Ca ratios in the teeth of dolphins represent a salinity gradient. The main aim was to determine whether these elements can be used as a natural tag for different aquatic environments. Teeth from 2 freshwater dolphins (Inia geoffrensis and Sotalia fluviatilis) and 2 marine species (S. guianensis and Pontoporia blainvillei) from Brazil and Uruguay were analyzed using a Laser Ablation Inductively Coupled Plasma-Mass Spectrometer. Intensity ratios of 138 Ba/ 43 Ca and 86 Sr/ 43 Ca were measured along a line that covered all growth increments in the dentin from the second year of life onwards. Teeth from the freshwater species had mean Ba/Ca values tenfold higher than marine dolphins, confirm- ing the inverse relationship between salinity (and thus ambient Ba/Ca) and elemental ratios in teeth. Furthermore, Ba/Ca ratios could also differentiate dolphins from lower-salinity estuarine areas from those in areas with minimal freshwater discharge. No significant differences were found for Sr/Ca values. Results presented encouraging indications for the application of this tech- nique as a potential new tool for studying habitat use in aquatic mammals.

Until the recent construction of hydroelectric dams, a series of 18 rapids divided the upper and lower Madeira River, and these rapids were thought to separate two species of Amazonian freshwater dolphins (boto): Inia boliviensis (above) and I. geoffrensis (below). Some reports and articles, however, mention the occurrence of botos within the rapids region and that they occasionally cross the rapids, but without mentioning the species concerned. Based on our previous studies, it is likely that I. boliviensis occurs in the region of the rapids. To test this supposition, we sampled 18 individuals from this region, and collected mitochondrial (control region, cytochrome b and cytochrome oxidase I) and nuclear (10 microsatellite loci) DNA data, in order to test if there is connectivity between the dolphins that were found within the rapids region and dolphins collected upstream and downstream of the rapids, and investigate population structuring between these localities. All animals in our study were molecularly identified using three mitochondrial markers as belonging to the species I. boliviensis. Animals upstream of the Teotonio waterfall, the main and highest waterfall of the region, had nuclear genome of I. boliviensis, while most dolphins downstream of the waterfall had nuclear genome of I. geoffrensis. Inia boliviensis collected in the rapids region above the Teotonio waterfall belong to a management unit (MU) distinct from the I. boliviensis MU occupying Bolivian rivers. Downstream of Teotonio waterfall most dolphins are I. boliviensis/geoffrensis hybrids, with remaining individuals being migrant I. boliviensis. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114, 764–777.

The aims of this study were to validate an enzyme immunoassay (EIA) for the measurement of luteinizing hormone (LH) in urine samples of Amazonian manatees (Trichechus inunguis; Mammalia: Sirenia) and to monitor urinary LH and reproductive steroids during the ovarian cycle in this species. Urine samples were collected from two captive males following a hormonal challenge with a gonadotropin-releasing hormone (GnRH) analogue. The urinary LH results from hormonal challenge were compared with urinary androgens for the purpose of EIA validation. Furthermore, urine samples were collected daily, over a 12-wk period, from two captive adult females, for 2 consecutive yr. The urinary LH pattern from females was compared with the patterns of urinary progestagens and estrogen conjugates throughout the ovarian cycle. An LH peak was observed in both male Amazonian manatees after the hormonal challenge, occurring prior to or together with peak androgen levels. In the females, the ovarian cycle ranged from 40 to 48 days (mean of 43.7 days). Two distinct peaks of estrogen conjugates were observed across all cycles analyzed, and the urinary LH peaks observed were accompanied by peaks of urinary estrogen conjugates. The EIA was validated as a method for the quantification of urinary LH from Amazonian manatees, as it was able to detect variations in the levels of LH in urine samples. These results suggest that T. inunguis exhibits a peculiar hormonal pattern during the ovarian cycle. Therefore, further studies are desirable and necessary to clarify the relationship between this hormonal pattern and morphological changes, as well as mating behavior, in Amazonian manatee.

Toothed whales have evolved to live in extremely different habitats and yet they all rely strongly on echolocation for finding and catching prey. Such biosonar based foraging involves distinct phases of searching for, approaching, and capturing prey, where echolocating animals gradually adjust sonar output to actively shape the flow of sensory information. Measuring those outputs in absolute levels requires hydrophone arrays centred on the biosonar beam axis, but this has never been done for wild toothed whales approaching and capturing prey. Rather, field studies make the assumption that toothed whales will adjust their biosonar in the same manner to arrays as they will when approaching prey. To test this assumption, we recorded wild botos (Inia geoffrensis) as they approached and captured dead fish tethered to a hydrophone in front of a star-shaped seven-hydrophone array. We demonstrate that botos gradually decrease interclick intervals and output levels during prey approaches, using stronger adjustment magnitudes than extrapolated from previous boto array data. Prey interceptions are characterised by high click rates, but although botos buzz during prey capture, they do so at lower click rates than marine toothed whales, resulting in a much more gradual transition from approach phase to buzzing. We also demonstrate for the first time that wild toothed whales broaden biosonar beamwidth when closing in on prey, as it is also seen in captive toothed whales and in bats, thus resulting in a larger ensonified volume around the prey, likely aiding prey tracking by decreasing the risk of prey evading ensonification.

Negative interactions between fishers and the Amazon River dolphin (Inia geoffrensis), or boto, have increased substantially in the last few decades. Herein, we investigate these interactions with focus on assessing fisher perceptions, attitudes, and behaviors toward botos. Moreover, we evaluate the effect that the Mamiraua Sustainable Development Reserve (MSDR) in the Brazilian Amazon, and related programs, has had on fisher attitudes and behaviors toward botos. The results suggest that interactions between fishers and botos, such as depredation and incidental entanglement, are frequent, and that the illegal harvest for botos, for use as bait, occurs in the majority of the study communities. However, the assessment revealed that most fishers have positive attitudes toward botos and that these attitudes have been influenced by participation in the MSDR activities such as research and ecotourism. Our results also highlight the importance of community-based enforcement in addressing the issue of boto harvesting. The MSDR programs have successfully promoted positive attitudes toward botos and have likely played a role in limiting boto mortality through behavioral controls, though the scope of influence of these programs has been restricted to a small geographical area. The current extent of these programs is insufficient to prevent the decline of the boto population; therefore, we suggest the MSDR model be used to improve and expand boto conservation efforts with communities in the region.

The mammaries from carcasses of two female Amazonian manatees were examined. Trichechus inunguis possesses two axillary mammaries beneath the pectoral fins, one on each side of the body. Each papilla mammae has a small hole on its apex--the ostium papillare. The mammaries are covered by a stratified squamous keratinized epithelium. The epithelium of the mammary ducts became thinner more deeply in the tissue and varied from stratified to simple cuboidal. There was no evidence of glandular activity or secretion into the ducts of the mammary glands.

The Negro River currently has seven floating houses where tourists can feed and interact with botos, each with its own history of how these aggregations were formed. Some keepers say these groups are familial, even reporting individuals being born into the group. However, behavioral studies have shown that botos are solitary, only forming groups at feeding areas and during the mating season. In the present study we used 12 microsatellite and molecular sex markers to characterize relationships within and between two boto aggregations (ten and seven botos each) in the lower Negro River. Molecular sexing revealed that all botos sampled from both aggregations were males. This may be explained by habitat preference, as male botos are primarily found in the main channels of large rivers, whereas females prefer more protected areas, such as flooded forests and its channels and lakes. Most of the animals were unrelated within each aggregation, demonstrating that these aggregations are not normally formed due to kinship bonds, but are exclusively for feeding, as botos learn that these places provide easy access to food. This study provides important information that helps us understand how human interaction is affecting the social structure and behavior of these animals. © 2019 PeerJ Inc.. All rights reserved.

The Amazonian manatee (Trichechus inunguis; Mammalia: Sirenia), a threatened aquatic mammal endemic to the Amazon basin, is the only sirenian that lives exclusively in fresh water. Information about the reproductive endocrinology of the Amazonian manatee is scarce; therefore, the aim of this study was to monitor salivary progesterone and estradiol patterns during the ovarian cycle in T. inunguis. Salivary samples were collected daily during a 12-week period of two consecutive years from two captive adult females. The salivary estradiol and progesterone were measured by enzyme immunoassay. The results were analyzed in an iterative process of excluding values that were higher than the mean plus 2 standard deviations until the basal values were determined. The interval between two peaks of salivary estradiol followed by a rise of progesterone was considered as one complete cycle for the calculation of the cycle length. We observed only three complete cycles in all samples analyzed. The cycle length ranged from 42 to 48 days (mean of 44.67 days). We also observed two distinct salivary estradiol peaks during all cycles analyzed, with the first peak occurring before the rise in salivary progesterone and the second occurred followed by a return to basal progesterone levels. This is the first in-depth study of the ovarian cycle in Amazonian manatees. Our results demonstrate that salivary samples can be a useful tool in the endocrine monitoring of this species and suggest that T. inunguis shows a peculiar hormonal pattern during the ovarian cycle, a finding that may have physiological and ecological significance in the reproductive strategy of these animals.

The Amazon River dolphin ( Inia geoffrensis ), or boto, is illegally harvested for use as bait in fisheries for the catfish Calophysus macropterus . To determine the effect of this harvest, we estimated apparent survival for a boto population in the central Brazilian Amazon where direct harvest is known to have occurred since 2000. For our analysis, we used capture and recapture/resighting data of 528 marked botos over a 17-year period (1994–2011). Time-dependent models estimated that apparent survival after the first reports of harvest ( ϕ = 0.899; SE = 0.007) was significantly lower than in years prior to harvest ( ϕ = 0.968; SE = 0.009). The decline in apparent survival suggests that current harvest rates exceed conservation limits and may be unsustainable. This issue requires the attention of natural resource managers from all countries of the Amazon basin, as the harvest is widespread and decline in survival could be mirrored in numerous locales.

During 2005-2007, a series of interviews were carried out with the riverside populations of the Oil Province of Urucu (AM, Brasil). The main goal of this work was to characterize the knowledge of the inhabitants of this area regarding the Amazonian manatee (Trichechus inunguis) (listed as Vulnerable on the IUCN Red List). Interviews were conducted with 74 local inhabitants from 33 communities. Between 2004 and 2007, 20 poached or entangled Amazonian manatees were reported in the area. Poaching with harpoon is the principal cause of mortality, but manatees are also incidentally caught in fishing nets. Interviewers described details of manatee hunting techniques in the area. The trade of manatee meat, although currently forbidden by Brazilian laws, still occurs in the area. Data obtained from the interviews suggest a dietary preference of the manatee for at least 29 species of aquatic and semi-aquatic plants. The interviewed fishermen showed a sound knowledge of Amazonian manatee biology and behavior. This information may be important to assist future conservation plans in the Amazon. © 2013 ABECO.

Classical and molecular cytogenetic (18S rDNA, telomeric sequence, and LINE-1 retrotransposon probes) studies were carried out to contribute to an understanding of the organization of repeated DNA elements in the Amazon River dolphin (boto, Inia geoffrensis). Twenty-seven specimens were examined, each presenting 2n = 44 chromosomes, the karyotype formula 12m + 14sm + 6st + 10t + XX/XY, and fundamental number (FN) = 74. C-positive heterochromatin was observed in terminal and interstitial positions, with the occurrence of polymorphism. Interstitial telomeric sequences were not observed. The nucleolar organizer region (NOR) was located at a single site on a smallest autosomal pair. LINE-1 was preferentially distributed in the euchromatin regions, with the greatest accumulation on the X chromosome. Although the karyotype structure in cetaceans is considered to be conserved, the boto karyotype demonstrated significant variations in its formula, heterochromatin distribution, and the location of the NOR compared to other cetacean species. These results contribute to knowledge of the chromosome organization in boto and to a better understanding of karyoevolution in cetaceans.

Sirenians have dichromatic colour vision and tactile hairs but have not developed underwater echolocation. Amazonian manatees (Trichechus inunguis) live in turbid water and it has been unclear how they understand their surroundings. In this study, we recorded the 3D movements of two captive-raised Amazonian manatees. The results revealed that the manatees always swam in a circular pattern. Both animals used slower, narrower turning motions as they approached the flooded forests, which is abundant in aquatic vegetation. Therefore, we suggest that these two manatees swam in a circular pattern to detect all directions of their surroundings especially using sensitive facial bristles.

Background: Establishment of reference values for serum biochemical analytes is important for monitoring health and physiological status of captive animals. Objective: The purpose of this study was to measure and report ranges for serum biochemical analytes in Amazonian manatees (Trichechus inunguis). Methods: Blood samples were collected from 24 healthy captive Amazonian manatees that comprised a mixture of adults, subadults, and calves and males and females; serum analytes were measured and analyzed using a dry reagent bench-top chemical analyzer. Comparisons were made between sexes and with previously published values of closely related species. Results: Medians and ranges (minimum–maximum) of values for the analytes were: lactate dehydrogenase (LDH), 151 (111–278) U/L (n=20); creatine kinase, 144 (76–315) U/L (n=11); alanine aminotransferase, 10 (2–28) U/L (n=18); aspartate aminotransferase, 14 (5–28) U/L (n=21); γ-glutamyltransferase, 47 (36–73) U/L (n=21); amylase, 1428 (1010–1874) U/L (n=21); alkaline phosphatase, 73 (36–141) U/L (n=19); total protein, 6.8 (6.2–8.0) g/dL (n=24); albumin, 3.3 (2.6–4.1) g/dL (n=21); cholesterol, 188 (101–399) mg/dL (n=21); triglycerides, 126 (60–236) mg/dL (n=21); glucose, 47 (22–69) mg/dL (n=21); urea, 43 (21–69) mg/dL (n=21); uric acid, 1.1 (0.5–1.8) mg/dL (n=22); creatinine, 2.2 (1.5–3.3) mg/dL (n=22); total bilirubin, 0.2 (0.2–2.0) mg/dL (n=21); calcium, 12.7 (10.2–18.6) mg/dL (n=24); iron, 282 (207–457) μg/dL (n=13); and magnesium, 6.9 (4.3–8.9) mg/dL (n=20). With the exception of LDH, no differences were observed between sexes. Conclusions: The ranges obtained in this study provide important preliminary estimates for concentrations and activities of serum analytes in Amazonian manatees until a larger reference interval study can be conducted.

Obligate river dolphins occur only in the rivers of Asia and South America, where they are increasingly subject to damaging pressures such as habitat degradation, food competition and entanglement in fishing gear as human populations expand. The Amazon basin hosts two, very different, dolphins—the boto or Amazon river dolphin (Inia geoffrensis) and the smaller tucuxi (Sotalia fluviatilis). Both species have wide geographical ranges and were once considered to be relatively abundant. Their IUCN Red List conservation status of Data Deficient (DD), due to limited information on threats, ecology, population numbers and trends, did not initially cause alarm. However, the development of dolphin hunting to provide fish bait at around the beginning of this millennium broadly coincided with the onset of a widespread perception that numbers of both species were in decline. Consequently, the need for population trend data to inform conservation advice and measures became urgent. This paper presents a 22-year time series of standardised surveys for both dolphins within the Mamirauá Reserve, Amazonas State, Brazil. Analysis of these data show that both species are in steep decline, with their populations halving every 10 years (botos) and 9 years (tucuxis) at current rates. These results are consistent with published, independent information on survival rates of botos in this area, which demonstrated a substantial drop in annual survival, commencing at around the year 2000. Mamirauá is a protected area, and is subject to fewer environmental pressures than elsewhere in the region, so there is no reason to suspect that the decline in dolphins within the Reserve is more pronounced than outside it. If South America’s freshwater cetaceans are to avoid following their Asian counterparts on the path to a perilous conservation status, effective conservation measures are required immediately. Enforcement of existing fishery laws would greatly assist in achieving this. © 2018 F. da Silva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

This study presents an overview of the appropriateness of the use of Amazonian cetaceans as sentinel species for environmental contamination by micropollutants, such as organochlorines and mercury. Due to the top position in food webs occupied by cetaceans and to their long life-span, high micropollutant concentrations have been verifi ed in their tissues, what allows an amplifi cation of the determination capacity by different analytical procedures. Besides providing an analytical advantage, the continuous micropollutant determination in cetaceans has been useful: as a reference of pollutant availability to other organisms; to integrate a complex signal of pollution; to quantify the ecological signifi cance of a contamination. Therefore, published information on the concentrations of the quoted pollutants in the Amazonian cetaceans Inia geoffrensis (boto) and Sotalia fl uviatilis (tucuxi) were gathered herewith. Carrying out comparison with data from Brazilian coastal cetaceans, it became clear how high the levels observed in Amazonian aquatic mammals are. In this context, mercury deserves to be highlighted. Despite the little volume of information on the concentrations of mercury, polychlorinated biphenyls and dichlorodiphenyltrichloroethane in Amazonian cetaceans, the existing information already demonstrates that the levels observed mirror the environmental contamination by such pollutants in distinct Amazonian environments. Perspectives for future investigations are also discussed, including possible ways of overcoming obstacles related to sampling of Amazonian cetaceans. Keywords: Brazil, Amazon, cetacean, mercury, organochlorines.

The Amazonian manatee (Trichechus inunguis) is uniparous and has a slow reproduction cycle due to a long gestation period and long interval between births. Even though protected by law, hunting remains one of the main causes hindering the natural population growth of this species in the wild. The histology and reproductive anatomy provide information on the history and reproductive status of the female and offer a tool for the conservation of the species. The present study describes the anatomy of the female reproductive tract in T. inunguis. It is based on materials from three reproductive tracts fixed in 10% buffered formalin. The ovaries, uterine tubes, uterus, vagina, and external genitalia are described. The hymen presents two tiny openings separated by a segment that, upon rupturing during the first copulation, should make up a single vaginal opening. A still intact hymen and the absence of placental scars in the uterus were found in one specimen. Additionally, the presence of a hemorrhagic body and Graafian follicles on the right ovary were observed, as well as whitish scars and among them, possible corpora albicantia. These findings suggest that T. inunguis undergoes infertile estrus cycles before its first gestation. Macroscopically, counting of the whitish scars is hindered by the small diameter of these structures. It is not possible to differentiate between the scars resulting from ruptured (corpora albicantia) and nonruptured follicles (regressed corpora atretica). The presence of whitish scars on both ovaries of the same specimen suggests their bilateral function in T. inunguis.

Toothed whales produce echolocation clicks with source parameters related to body size; however, it may be equally important to consider the influence of habitat, as suggested by studies on echolocating bats. A few toothed whale species have fully adapted to river systems, where sonar operation is likely to result in higher clutter and reverberation levels than those experienced by most toothed whales at sea because of the shallow water and dense vegetation. To test the hypothesis that habitat shapes the evolution of toothed whale biosonar parameters by promoting simpler auditory scenes to interpret in acoustically complex habitats, echolocation clicks of wild Amazon river dolphins were recorded using a vertical seven-hydrophone array. We identified 404 on-axis biosonar clicks having a mean SLpp of 190.3±6.1 dB re. 1 µPa, mean SLEFD of 132.1±6.0 dB re. 1 µPa2s, mean Fc of 101.2±10.5 kHz, mean BWRMS of 29.3±4.3 kHz and mean ICI of 35.1±17.9 ms. Piston fit modelling resulted in an estimated half-power beamwidth of 10.2 deg (95% CI: 9.6–10.5 deg) and directivity index of 25.2 dB (95% CI: 24.9–25.7 dB). These results support the hypothesis that river-dwelling toothed whales operate their biosonars at lower amplitude and higher sampling rates than similar-sized marine species without sacrificing high directivity, in order to provide high update rates in acoustically complex habitats and simplify auditory scenes through reduced clutter and reverberation levels. We conclude that habitat, along with body size, is an important evolutionary driver of source parameters in toothed whale biosonars.

Odontoceti emit broadband high-frequency clicks on echolocation for orientation or prey detection. In the Amazon Basin, two odontoceti species, boto (Amazon River dolphin, Inia geoffrensis) and tucuxi (Sotalia fluviatilis), live sympatrically. The acoustic characteristics of the echolocation clicks of free-ranging botos and tucuxis were measured with a hydrophone array consisting of a full-band and an acoustic event recorder (A-tag). The clicks of the two species were short-duration broadband signals. The apparent source level was 201 dB 1 μPa peak-to-peak at 1 m in the botos and 181 dB 1 μPa peak-to-peak at 1 m in the tucuxis, and the centroid frequency was 82.3 kHz in the botos and 93.1 kHz in the tucuxis. The high apparent source level and low centroid frequency are possibly due to the difference in body size or sound production organs, especially the nasal structure, the sound source of clicks in odontoceti.

We used mitochondrial DNA control region sequences to examine phylogeography and population differentiation of the endangered Amazonian manatee Trichechus inunguis. We observe lack of molecular differentiation among localities and we find weak association between geographical and genetic distances. However, nested clade analysis supports restricted gene flow and/or dispersal with some long-distance dispersal. Although this species has a history of extensive hunting, genetic diversity and effective population sizes are relatively high when compared to the West Indian manatee Trichechus manatus. Patterns of mtDNA haplotype diversity in T. inunguis suggest a genetic disequilibrium most likely explained by demographic expansion resulting from secession of hunting and enforcement of conservation and protective measures. Phylogenetic analysis of T. manatus and T. inunguis haplotypes suggests that T. inunguis is nested within T. manatus, effectively making T. manatus a paraphyletic entity. Paraphyly of T. manatus and recent divergence times of T. inunguis and the three main T. manatus lineages suggest a possible need for a taxonomic re-evaluation of the western Atlantic Trichechus.

Oceanic dolphins (Odontoceti: Delphinidae) produce tonal whistles, the structure and function of which have been fairly well characterized. Less is known about the evolutionary origins of delphinid whistles, including basic information about vocal structure in sister taxa such as the Platanistidae river dolphins. Here we characterize vocalizations of the Amazon River dolphin (Inia geoffrensis), for which whistles have been reported but not well documented. We studied Inia at

In the present study we tested if a series of 18 rapids on the upper Madeira River form an effective barrier to gene flow, and in particular if they delimit the distribution of the boto Inia boliviensis—which it is believed to occurs only in the Bolivian sub-basin, above the rapids—and I. geoffrensis, which occurs throughout the Amazon basin and below the upper Madeira River rapids. We analyzed 125 individuals from the Madeira River basin sampled from upstream and downstream of the rapids. As the two species are morphologically similar, we used diagnostic molecular characters from known reference specimens to assign individuals to species. We observed that all individuals of Inia from the Bolivian sub-basin up to almost the mouth of the Madeira River belong to the species I. boliviensis. Therefore we concluded that the rapids do not delimit the distribution of I. boliviensis upstream and I. geoffrensis downstream of the rapids as previously hypothesized. Since we registered I. boliviensis along almost the entire length of the Madeira River, we estimated gene flow, time of divergence and effective population sizes of the upstream (Bolivian) and downstream (Madeira River) groups of I. boliviensis using IMa2. We concluded that gene flow is uni-directional from the upstream to the downstream group. Divergence time between the two groups was estimated to have occurred ~122 thousand years ago. The coalescent effective population size for the upstream group was estimated at ~131 thousand individuals, while for the downstream group it was estimated at ~102 thousand individuals. Recently two dams have been constructed in the region of the rapids; neither has a mechanism that will maintain connectivity between the upstream and downstream regions, and together with anthropogenic alterations to the hydrodynamic regime and ecology of the river will likely pose serious long-term and short-term consequences for I. boliviensis and other aquatic taxa.

True river dolphins are some of the rarest and most endangered of all vertebrates. They comprise relict evolutionary lineages of high taxonomic distinctness and conservation value, but are afforded little protection. We report the discovery of a new species of a river dolphin from the Araguaia River basin of Brazil, the first such discovery in nearly 100 years. The species is diagnosable by a series of molecular and morphological characters and diverged from its Amazonian sister taxon 2.08 million years ago. The estimated time of divergence corresponds to the separation of the Araguaia-Tocantins basin from the Amazon basin. This discovery highlights the immensity of the deficit in our knowledge of Neotropical biodiversity, as well as vulnerability of biodiversity to anthropogenic actions in an increasingly threatened landscape. We anticipate that this study will provide an impetus for the taxonomic and conservation reanalysis of other taxa shared between the Araguaia and Amazon aquatic ecosystems, as well as stimulate historical biogeographical analyses of the two basins. © 2014 Hrbek et al.

In this study we report the hematological, biochemical and hormonal parameters in a juvenile male Amazonian manatee measured before transport, immediately after transport, and during adaptation to a new facility. The animal was transported from Manaus, Amazonas State, Brazil, to Sao Paulo, Sao Paulo State, Brazil, (2,733 km) within 6 hours. Among all blood parameters analyzed, we observed obvious neutrophilia, lymphopenia, and increases in the neutrophil/lymphocyte ratio and serum glucose and aspartate aminotransferase (AST) levels, but these parameters subsequently returned to normal. These results suggest that transport and changes in the environment are temporary stressful events for Amazonian manatees. We, therefore, recommend monitoring the hematological and biochemical parameters before and after translocation to minimize the effects of handling stressors in this species.

The Amazonian manatee (Trichechus inunguis) is a threatened aquatic mammal endemic to the Amazon basin. The aim of this study was to evaluate the urinary and salivary reproductive hormone levels of captive Amazonian manatees collected during two seasons of the year. Salivary samples from four males and urinary and salivary samples from three females were collected during two seasons (March-June and September-November) over two consecutive years.Salivarytestosteroneinmaleswasmeasuredbyradioimmunoassayandreproductivehormonesinfemales(salivary progesterone and oestradiol and urinary progestogens, oestrogens and luteinising hormone) were measured by enzyme immunoassay. The data were analysed in a 2� 2 factorial design, where the factors were year and season. There was no effect of year or season for salivary testosterone. All female hormones showed a seasonal effect (higher hormone levels during March-June than September-November) or an interaction between year and season (P,0.05). These results strongly indicate the existence of reproductive seasonality in Amazonian manatees; however, apparently only females exhibit reproductive quiescence during the non-breeding season. Further long-term studies are necessary to elucidate which environmental parameters are related to reproductive seasonality in T. inunguis and how this species responds physiologically to those stimuli. Additional keywords: reproduction, saliva, sirenian, steroids, urine.

We present a description of osteological alterations observed in the tucuxi, Sotalia fluviatilis (Gervais, 1853) from a sample of 43 specimens. Fractures were the most frequent alterations in the sample (16%), occurring in various regions of the skeleton such as the ribs, hyoid apparatus, transverse and neural processes of vertebrae and scapula. We observed three individuals with ankylosis between the cervical vertebrae and two individuals with morphological changes (cranio-caudally elongated hemal arch and flattened cranial margin of the scapula). The only observed pathology was a case of osteomyelitis in the left dentary, which caused the loss of teeth, deformation of the associated alveoli and the formation of a medial fistula (lingual) for drainage of purulent material. This represents the first record of osteomyelitis in S. fluviatilis.

Com o objetivo de testar o tempo de passagem do alimento no trato digestório de peixes-bois da Amazônia em cativeiro, foram testadas separadamente duas dietas distintas. Uma composta exclusivamente de capim do gênero Brachiaria (dieta experimental - DE 1) e outra de capim do gênero Brachiaria acrescentado de pequenas porções de ração extrusada para eqüinos (dieta experimental - DE 2). Foram selecionados do plantel do INPA dois animais adultos machos sadios, os quais foram isolados dos demais e submetidos a um período de aclimatação às dietas experimentais por 15 dias. Após este período, as dietas foram marcadas com uma fita plástica de 10 cm e fornecidas aos animais que foram monitorados em intervalos de uma hora. Todo material fecal foi coletado até a recuperação dos marcadores plásticos. A média do tempo de passagem da DE 1 foi de 123h57min, cerca de 5,15 dias e da DE 2 foi de 125h04min ou 5,21 dias. Não houve diferença estatística (P

Studies of the feeding behaviour of aquatic species in their natural environment are difficult, since direct observations are rarely possible. In this study, a newly developed animal-borne underwater sound recorder (AUSOMS-mini) was applied to captive Amazonian (Trichechus inunguis) and Antillean (Trichechus manatus manatus) manatees in order to directly record their feeding sounds. Different species of aquatic plants were offered to the manatees separately. Feeding sounds were automatically extracted using a custom program developed with MATLAB. Compared to ground truth data, the program correctly detected 65–79% of the feeding events, with a 7.3% or lower false alarm rate, which suggests that this methodology is a useful recorder of manatee feeding events. All manatees foraged during both the daytime and night-time. However, manatees tended to be less active and masticated slower during the night than during the day. The manatee mastication cycle duration depended on plant species and individual. This animal-borne acoustic monitoring system could greatly increase our knowledge of manatee feeding ecology by providing the exact time, duration and number of feeding events, and potentially the plant species being fed on.

Previous studies suggested that manatee calls were primarily for communication and not for navigational purposes. In this study, the vocal response of captive Amazonian manatees to playbacks of several acoustic stimuli was investigated. Experiments were conducted using nine captive Amazonian manatees at the National Institute of Amazonian Research (INPA), Brazil, in 2014. All manatees, except one, are orphan calves rescued from the illegal hunting or incidental catch. They were kept in the outdoor pool with a group of 2-3 individuals, which are considered to be related. Manatees were exposed to five different playback stimuli: a recorded vocal from a related manatee, a recorded vocal from an unrelated manatee, a synthesized constant frequency based on the fundamental frequency of a related manatee vocal, a synthesized sound which entirely different from manatee vocal, and silence. A total of 58 playback sessions was conducted and 22,590 calls were recorded. While manatees showed inter-individual variabili...