CHARACTERIZATION OF THE FUNGAL MICROBIOTA IN THE NOSTRILS AND RECTUM OF AMAZONIAN MANATEES (TRICHECHUS INUNGUIS) FROM A REHABILITATION PROGRAM IN BRAZIL.

The present study characterized the filamentous and yeast-like fungal microbiota of the nasal cavity and rectum of Amazonian manatees (Trichechus inunguis) undergoing rehabilitation at the Laboratory of Aquatic Mammals, National Institute of Amazonian Research, Manaus, Amazonas, and determined the antifungal susceptibility of these organisms. Nasal and rectal swabs were collected from 22 calves and three juveniles. The samples were seeded in Sabouraud agar supplemented with chloramphenicol 10%, incubated at 26°C, and observed daily for up to 7 d. The growth of different filamentous and yeast-like fungi was observed among the two anatomical sites. Filamentous fungi were categorized by macro- and microscopic characteristics of the colonies. Representatives of each group were selected for molecular identification based on the internal transcribed spacer region. Yeast identification was performed using MALDI-TOF MS and molecular analyses. Thirteen genera of filamentous fungi and six genera of yeasts were isolated and identified. The dominant filamentous species were Fusarium spp., Aspergillus spp., and Cochliobolus lunatus in the nostril samples and Aspergillus melleus in the rectal samples. Candida was the dominant genus among the identified yeasts at both anatomical sites. In the antifungal susceptibility test, 28 isolates showed resistance to fluconazole (78%), itraconazole (39%), and nystatin (42%). The knowledge of fungal microbiota composition of Amazonian manatees provides information that assists in monitoring the health status of individuals maintained in captivity, as these organisms can behave either as opportunists or as primary pathogens. Moreover, the composition and resistance of these organisms may vary among different rehabilitation institutions or different time frames of search, reinforcing the importance of constant in loco surveillance of these microorganisms. This study provides new perspectives on the fungal diversity in the microbiota of manatees and supports future studies concerning the clinical and epidemiological aspects and the impacts of these agents on the health of Amazonian manatees undergoing rehabilitation.

Virulence factors of Gram-negative bacteria from free-ranging Amazon river dolphins (Inia geoffrensis).

Freshwater cetaceans play a significant role as sentinel animals, providing important data on animal species and aquatic ecosystem health. They also may serve as potential reservoirs of emerging pathogens and host virulence genes in their microbiota. In this study, we evaluated virulence factors produced by Gram-negative bacteria recovered from individuals belonging to two populations of free-ranging Amazon river dolphins (Inia geoffrensis). A total of 132 isolates recovered from the oral cavity, blowhole, genital opening and rectum of 21 river dolphins, 13 from Negro River and 8 from Tapajós River, Brazil, were evaluated for the production of virulence factors, such as biofilms and exoproducts (proteases, hemolysins and siderophores), in planktonic and biofilm forms. In planktonic form, 81.1% (107/132) of the tested bacteria of free-ranging Amazon river dolphins were able to produce virulence factors, with 44/132 (33.4%), 65/132 (49,2%) and 54/132 (40,9%) positive for protease, hemolysin and siderophore production, respectively. Overall, 57/132 (43.2%) of the isolates produced biofilms and, under this form of growth, 66/132 (50%), 88/132 (66.7%) and 80/132 (60.6%) of the isolates were positive for protease, hemolysin and siderophore production. In general, the isolates showed a higher release of exoproducts in biofilm than in planktonic form (P < 0.001). The present findings show that Amazon river dolphins harbor potentially pathogenic bacteria in their microbiota, highlighting the importance of monitoring the micro-organisms from wild animals, as they may emerge as pathogens for humans and other animals.

Hemotropic Mycoplasma spp. in Aquatic Mammals, Amazon Basin, Brazil.

Hemotropic Mycoplasma spp. (hemoplasmas) are uncultivable bacteria that infect mammals, including humans. We detected a potentially novel hemoplasma species in blood samples from wild river dolphins in the Amazon River Basin, Brazil. Further investigation could determine pathogenicity and zoonotic potential of the detected hemoplasma.

One Health Implications of Antimicrobial Resistance in Bacteria from Amazon River Dolphins.

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 Tapajós 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 Tapajós 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 < 0.001). The results indicate that free-ranging Amazon river dolphins host resistant bacteria, contributing for their maintenance in the environment. This study highlights the importance of the One Health approach to monitor the emergence of antimicrobial resistance. Summary Gram-negative bacteria recovered from 21 free-ranging Amazon river dolphins (Inia geoffrensis) from the Negro River and the Tapajós River populations were evaluated for their antimicrobial susceptibility. 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. Thus, free-ranging Amazon river dolphins, never treated with antimicrobials, host resistant bacteria, contributing for their maintenance in the environment and highlighting the importance of the One Health approach to monitor the emergence of antimicrobial resistance.

Hematologic profile of Amazon river dolphins Inia geoffrensis and its variation during acute capture stress.

Hematological values are of primary importance when investigating the health and physiological status of populations as they reflect the biological equilibrium of aquatic ecosystems. The objectives of this study are to produce baseline values for hematological parameters of the Amazon River dolphin (Inia geoffrensis), as well as to investigate significant variations according to sex, age, reproductive status and stress level. One-hundred-and-ten dolphins from Mamirauá Sustainable Development Reserve (3°3'S, 64°51'W), Central Amazon, Brazil, were live captured and sampled in November 2004 and 2005. Further, the means, standard deviations, minimum and maximum values and reference values (90% CI) were calculated. Correlations were performed to assess the relationships among blood values and cardiac rate (CR), respiratory frequency (RF), handling time and level of stress. No significant differences were found between sexes. Also, no differences occurred among pregnant and non-pregnant females, pregnant females and adult males or non-pregnant females and adult males. Calves had a higher white blood cell (WBC) count, and the neutrophil and lymphocyte absolute counts were significantly higher in calves than adults. The level of stress determined by empirical observation positively correlated with the WBC, neutrophil, lymphocyte and monocyte absolute counts and CR and RF. It was found that less stressed animals tend to present lower platelet counts and lower CR. The handling time of the dolphins was positively correlated with hematocrit (Hct), red blood cells (RBC) and Hb level. The hematological and physiological parameters varied according to time of handling and proved to be a good bioindicator of acute stress in Amazon River dolphins. The data provided here can complement long-term monitoring and identify the early warning indicators of health problems at the population level.

Both cetaceans in the Brazilian Amazon show sustained, profound population declines over two decades.

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.

Cutaneous mycobacteriosis in a captive Amazonian manatee Trichechus inunguis.

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.

Morphology and Ultrastructure of the Amazon River Dolphin (Inia geoffrensis) Spermatozoa.

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.

Volumen ovárico y valores hormonales en síndrome de ovario poliquístico / Ovarian volume and hormone concentrations in polycystic ovary syndrome

Antecedentes: El síndrome de ovario poliquístico se caracteriza por hiperandrogenismo clínico y bioquímico, morfología de ovarios poliquísticos, alteración en la secreción de las gonadotropinas, resistencia a la insulina y/o hiperinsulinemia asociada a obesidad. Objetivo: El objetivo del presente estudio fue comparar las correlaciones entre los niveles hormonales de FSH, LH, estradiol, AMH, y el número de folículos antrales de las mujeres con síndrome de ovario poliquístico. Pacientes y métodos: Las pacientes se agruparon de acuerdo a la presencia de oligomenorrea y amenorrea, se determinaron los niveles de AHM, FSH, LH y estradiol, además se les realizó un ultrasonido transvaginal para determinar el volumen ovárico y el número de folículos antrales. Resultados: El análisis por correlación de Pearson reveló una correlación significativa (0,283, p = 0,01) entre la hormona antimulleriana y el número de folículos antrales, lo que demostró que a mayor número de folículos antrales mayores concentraciones de hormona antimulleriana. Mientras que la correlación lineal mostró que la hormona antimulleriana correlacionó positivamente con el número de folículos antrales (r = 0,303, p = 0,002). La FSH y LH correlacionaron de manera negativa con el número de folículos antrales en ambos grupos (r = -0,182, p = 0,05). El volumen ovárico correlacionó positivamente con el número de folículos (r = 0,708, p = 0,000). Conclusiones: Creemos que AHM puede ser utilizado como un marcador en conjunción con los valores de ultrasonido para evaluar la reserva ovárica en el futuro

Background: Polycystic ovary syndrome is characterized by clinical and biochemical hyperandrogenism, polycystic ovary morphology, altered gonadotropin secretion, insulin resistance, and/or hyperinsulinemia associated with obesity. Objective: To compare the correlations between hormone levels of FSH, LH, estradiol, AMH, and antral follicle numbers in women with polycystic ovary syndrome. Patients and methods: Patients were grouped according to the presence of oligomenorrhea and amenorrhea. Levels of AHM, FSH, LH and estradiol were determined and transvaginal ultrasound was performed to determine ovarian volume and the number of antral follicles. Results: Analysis by Pearson correlation revealed a significant correlation (0.283, p = .01) between AMH and the number of antral follicles: the higher the number of antral follicles, the higher the concentrations of AMH. Linear correlation showed that AMH levels were positively correlated with the number of antral follicles (r = 0.303, p = .002). FSH and LH were negatively correlated with the number of antral follicles in both groups (r = -0.182, p = .05). Ovarian volume was positively correlated with the number of follicles (r = 0.708, p = .000). Conclusions: We believe that AHM can be used as a marker in conjunction with ultrasound values to assess ovarian reserve in future

Acoustic characteristics of biosonar sounds of free-ranging botos (Inia geoffrensis) and tucuxis (Sotalia fluviatilis) in the Negro River, Amazon, Brazil.

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.

When You Get What You Haven't Paid for: Molecular Identification of "Douradinha" Fish Fillets Can Help End the Illegal Use of River Dolphins as Bait in Brazil.

The fishery for Calophysus macropterus, an Amazonian necrophagous catfish, is highly detrimental to river dolphins and caimans, which are deliberately killed for use as bait. In the Brazilian Amazon, this fishery has increased over the last decade, in spite of the rejection of scavenger fishes by Brazilian consumers. It was suspected that C. macropterus fillets were being sold in Brazilian markets, disguised as a fictitious fish (the "douradinha"). We collected 62 fillets from "douradinha" and other suspiciously named fish from 4 fish-processing plants sold at 6 markets in Manaus, in the Brazilian Amazon, and sequenced the cytochrome b gene to identify fillets to species. Sixty percent of fillets labeled "douradinha" or with other deceptive names were actually C. macropterus. Six other fish species of low commercial value were also found. The presence of dolphin tissue in the stomach contents of C. macropterus was confirmed by mtDNA control region sequencing. Our results formed the scientific basis for a moratorium on the fishing and fraudulent selling of C. macropterus, issued by the Brazilian Ministries of the Environment and Fisheries. Exposure of this fraud via the mass media can help end the illegal use of dolphins as bait in Brazil.

Efectos de la morfina en la plasticidad cerebral / Effects of morphine on brain plasticity

Introducción: La morfina, como otros opiáceos y las drogas de abuso, tiene la capacidad de modificar la plasticidad cerebral de las áreas que regulan la morfología neuronal de las dendritas y espinas, que son el sitio primario de sinapsis excitatorias en regiones cerebrales que regulan funciones de incentivo motivación, recompensa y aprendizaje. Objetivo: En la presente revisión se analizan aspectos del impacto del uso de la morfina durante los periodos prenatales del desarrollo cerebral y las consecuencias a largo plazo en murinos, para relacionar estos efectos que ocurren en el humano neonato y adulto. Desarrollo: La exposición repetida a la morfina en el tratamiento del dolor en enfermos terminales produce cambios a largo plazo en la densidad postsináptica de sitios (dendritas y espinas) en áreas sensibles del cerebro, como la corteza prefrontal y el sistema límbico (hipocampo, amígdala), así como en los núcleos caudado y accumbens. Este artículo revisa los mecanismos celulares implicados, principalmente de los receptores dopaminérgicos y glutamatérgicos, así como la plasticidad sináptica lograda por los cambios en las dendritas y espinas en esta área. Conclusiones: Las acciones de la morfina durante el desarrollo del cerebro y también en el cerebro adulto producen alteraciones en la plasticidad de sitios excitatorios postsinápticos, áreas del cerebro que están implicadas en las funciones del sistema límbico (la recompensa y el aprendizaje). Se necesitan más estudios sobre la plasticidad en las dendritas y espinas en sus moléculas de señalización, tales como el calcio, con el fin de mejorar el tratamiento de la adicción

Introduction: Morphine shares with other opiates and drugs of abuse the ability to modify the plasticity of brain areas that regulate the morphology of dendrites and spines, which are the primary sites of excitatory synapses in regions of the brain involved in incentive motivation, rewards, and learning. Objective: In this review we discuss the impact of morphine use during the prenatal period of brain development and its long-term consequences in murines, and then link those consequences to similar effects occurring in human neonates and adults. Development: Repeated exposure to morphine as treatment for pain in terminally ill patients produces long-term changes in the density of postsynaptic sites (dendrites and spines) in sensitive areas of the brain, such as the prefrontal cortex, the limbic system (hippocampus, amygdala), and caudate nuclei and nucleus accumbens. This article reviews the cellular mechanisms and receptors involved, primarily dopaminergic and glutamatergic receptors, as well as synaptic plasticity brought about by changes in dendritic spines in these areas. Conclusions: The actions of morphine on both developing and adult brains produce alterations in the plasticity of excitatory postsynaptic sites of the brain areas involved in limbic system functions (reward and learning). Doctors need further studies on plasticity in dendrites and spines and on signaling molecules, such as calcium, in order to improve treatments for addiction

Effects of morphine on brain plasticity.

INTRODUCTION: Morphine shares with other opiates and drugs of abuse the ability to modify the plasticity of brain areas that regulate the morphology of dendrites and spines, which are the primary sites of excitatory synapses in regions of the brain involved in incentive motivation, rewards, and learning. OBJECTIVE: In this review we discuss the impact of morphine use during the prenatal period of brain development and its long-term consequences in murines, and then link those consequences to similar effects occurring in human neonates and adults. DEVELOPMENT: Repeated exposure to morphine as treatment for pain in terminally ill patients produces long-term changes in the density of postsynaptic sites (dendrites and spines) in sensitive areas of the brain, such as the prefrontal cortex, the limbic system (hippocampus, amygdala), and caudate nuclei and nucleus accumbens. This article reviews the cellular mechanisms and receptors involved, primarily dopaminergic and glutamatergic receptors, as well as synaptic plasticity brought about by changes in dendritic spines in these areas. CONCLUSIONS: The actions of morphine on both developing and adult brains produce alterations in the plasticity of excitatory postsynaptic sites of the brain areas involved in limbic system functions (reward and learning). Doctors need further studies on plasticity in dendrites and spines and on signaling molecules, such as calcium, in order to improve treatments for addiction.

Seasonal variation in urinary and salivary reproductive hormone levels in Amazonian manatees (Trichechus inunguis).

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. Salivary testosterone in males was measured by radioimmunoassay and reproductive hormones in females (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.

Exposure to sublethal concentrations of copper changes biochemistry parameters in silver catfish, Rhamdia quelen, Quoy & Gaimard.

The effects of Cu exposure on catalase (CAT) and acetylcholinesterase (AChE) activity, formation of thiobarbituric acid-reactive species (TBARS) and metabolic parameters were evaluated in silver catfish (Rhamdia quelen). The fish were exposed for 45 days to 0, 16 and 29 µg/L Cu. The fish that were exposed to Cu exhibited lower TBARS levels in the muscle and higher TBARS levels in the liver. They also showed lower CAT activity in the liver and lower AChE activity in the brain and muscle. Higher glucose and lactate and lower protein plasma levels were observed in the fish exposed to Cu. The changes in the hepatic metabolic parameters were Cu concentration dependent. In the muscle, lower glycogen and higher lactate levels were observed in the fish exposed to Cu. Alterations in the metabolic parameters showed a preference for the anaerobic pathway of energy production and liver protein catabolism to supply the energy demand.

First attempt to monitor luteinizing hormone and reproductive steroids in urine samples of the Amazonian manatee (Trichechus inunguis).

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.

Endocrine monitoring of the ovarian cycle in captive female Amazonian manatees (Trichechus inunguis).

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.

Molecular cytogenetic characterization of the Amazon River dolphin Inia geoffrensis.

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.

Occurrence of antibodies to Toxoplasma gondii and Lepstospira spp. in manatees (Trichechus inunguis) of the Brazilian Amazon.

The presence of Toxoplasma gondii and Leptospira spp. antibodies was investigated in 74 manatees (Trichechus inunguis [Mammalia: Sirenia]) kept in captivity in two rescue units in the northern region of Brazil. Antibodies to T. gondii were detected in 29 (39.2%) of 74 animals by using the modified agglutination test (titer, 1:25). For antibodies against Leptospira spp., sera were diluted 1:50 and tested against 24 strains ofleptospires by microscopic agglutination microtechnique, and positive samples were end titrated. Twenty-three (31.1%) of 74 animals were reactive to four serovars (Patoc 21/23, Castellonis 2/23, Icterohaemorrhagiae 1/23, and Butembo 1/ 23), with titers ranging from 100 to 1,600. This is the first report of antibodies against T. gondii and Leptospira spp. in T. inunguis from the Brazilian Amazon.

Phylogenetic status and timescale for the diversification of Steno and Sotalia dolphins.

Molecular data have provided many insights into cetacean evolution but some unsettled issues still remain. We estimated the topology and timing of cetacean evolutionary relationships using bayesian and maximum likelihood analyses of complete mitochondrial genomes. In order to clarify the phylogenetic placement of Sotalia and Steno within the Delphinidae, we sequenced three new delphinid mitogenomes. Our analyses support three delphinid clades: one joining Steno and Sotalia (supporting the revised subfamily Stenoninae); another placing Sousa within the Delphininae; and a third, the Globicephalinae, which includes Globicephala, Feresa, Pseudorca, Peponocephala and Grampus. We also conclude that Orcinus does not belong in the Globicephalinae, but Orcaella may be part of that subfamily. Divergence dates were estimated using the relaxed molecular clock calibrated with fossil data. We hypothesise that the timing of separation of the marine and Amazonian Sotalia species (2.3 Ma) coincided with the establishment of the modern Amazon River basin.