Herpesvirus and adenovirus surveillance in threatened wild West Indian (Trichechus manatus) and Amazonian manatees (Trichechus inunguis), Brazil.

THE FAMILY TRICHECHIDAE (ORDER SIRENIA) COMPRISES THREE SPECIES: African (Trichechus senegalenses), West Indian (T. manatus), WIM)], and the Amazonian manatees (T. inunguis, AMM). Whereas WIM inhabits both riverine and coastal systems in the western Atlantic, AMM is the only exclusively freshwater sirenian, endemic to the Amazon River Basin. The study of infectious agents is essential to species conservation, especially considering that both species are classified as Vulnerable by the IUCN Red List and as Endangered by the Brazilian Red List. The current knowledge about viral agents in sirenians is scarce. Herpesviruses and adenovirus are DNA viruses able to infect and cause disease in a wide range of hosts. Herein, we used panPCR protocols to survey herpesvirus and adenovirus in blood samples of wild WIM (n = 23) and AMM (n = 26) under human care in Brazil. Herpesvirus DNA was detected in one juvenile female WIM (1/23; 4.3%; 95% CI -4.7 - 13.3) from Ceará state and in four AMM (two juvenile females, a juvenile male, and an adult female; 4/26; 15.4%; 95% CI 0.5 - 30.3) from Amazonas state. The two different gammaherpesvirus DNA polymerase sequence types identified (one per species, a sequence type in a WIM and another one in three AMM) were highly similar (99% nucleotide identity) to Trichechid herpesvirus 1, reported in West Indian manatees of Florida (USA), and 100% identical when translated into amino acids. A herpesviral glycoprotein B sequence was identified in two AMM. None of the samples was positive to adenovirus. To the authors' knowledge, this is the first herpesvirus detection in manatees from South America, expanding the herpesvirus geographical range, and the first in WIM and AMM worldwide. Our findings suggest (i) that West Indian and Amazonian manatees are possibly the natural hosts of the detected herpesvirus, and (ii) coevolution of that gammaherpesvirus with Trichechus. Future studies are necessary to characterize the obtained virus and elucidate potential pathological effects (if any) in these species.

Manatee calf call contour and acoustic structure varies by species and body size.

Vocal activity and signal characteristics of mammals are driven by several factors that result in both stability and plasticity over multiple time scales. All three extant species of manatee communicate with several calls that are especially important for maintaining contact between cows and calves. Determining if calf calls differ across manatee species will provide insights into the evolution of species-specific acoustic communication traits. We investigated the interspecific differences in the vocalizations of calves of Amazonian manatees (Trichechus inunguis) and the two subspecies of the West Indian manatee (T. manatus). Vocalizations of individual calves were recorded in rehabilitation centers in Brazil, Puerto Rico, the United States, and Mexico. The acoustic structure of calls produced by manatee calves varied between species and with body size. Amazonian manatee calves produced shorter calls with multiple notes at higher frequency while West Indian calves produced modulated calls that were lower in frequency and longer in duration. Smaller West Indian calves produced frequency modulated, hill-shaped calls that flattened with an increase in body length. Our results provide evidence for divergence in the ontogeny of vocalizations across T. manatus and T. inunguis and suggest variation in body size contributed to the evolution of differences in the characteristics of their calls.

Highly divergent herpesviruses in threatened river dolphins from Brazil.

River dolphins are a highly threatened polyphyletic group comprised of four odontocete families: Iniidae, Pontoporiidae, Lipotidae, and Platanistidae, the first two endemic to South America. To address the knowledge gap regarding infectious agents in this cetacean group, we surveyed the presence of herpesviruses by PCR in skin and/or blood samples of live-captured Amazon (Inia geoffrensis, n = 25) and Bolivian (Inia boliviensis, n = 22) river dolphins of the Amazon basin and in selected tissue samples of franciscanas (Pontoporia blainvillei, n = 27) stranded or bycaught in southeastern Brazil. Additionally, available franciscana tissue samples were examined by histopathology. Herpesvirus DNA was amplified in 13 Bolivian river dolphins (59.1%, 95% CI 38.5-79.6%) and 14 franciscanas (51.9%, 95% CI 33.0-70.7%). All Amazon river dolphins were herpesvirus-negative. Two different herpesviruses were found in Bolivian river dolphins: a previously known gammaherpesvirus detected in blood and/or skin samples of all positive individuals and a novel alphaherpesvirus in the skin of one animal. A new gammaherpesvirus was found in several franciscana samples-the first herpesvirus recorded in Pontoporiidae. Intranuclear inclusion bodies consistent with herpesvirus were observed in the lymph node of one franciscana. The high divergence among the obtained herpesviruses and those previously described can be explained by viral-host coevolution, and by the fact that these populations are fairly isolated.

Boto (Inia geoffrensis-Cetacea: Iniidae) aggregations in two provisioning sites in the lower Negro River-Amazonas, Brazil: are they related?

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.

Amazon river dolphins (Inia geoffrensis) modify biosonar output level and directivity during prey interception in the wild.

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 predicted 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 is also seen in captive toothed whales and bats, thus resulting in a larger ensonified volume around the prey, probably aiding prey tracking by decreasing the risk of prey evading ensonification.

The mammary glands of the Amazonian manatee, Trichechus inunguis (Mammalia: Sirenia): morphological characteristics and microscopic anatomy.

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.

A new species of river dolphin from Brazil or: how little do we know our biodiversity.

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.

Presumed normal ultrasonographic findings of the testis and epididymis of botos (Inia geoffrensis).

Fifteen live adult male botos, or Amazon river dolphins (Inia geoffrensis), were examined using ultrasonography during the yearly capture expedition, between October and November 2005, at the Mamirauá Sustainable Development Reserve, within the Brazilian Amazon (3 degrees S, 65 degrees W). All examinations were performed with a Sonosite 180 plus ultrasound unit in conjunction with a 2- to 5-MHz multifrequency transducer convex array 180 Plus/Elite-C60. Age and maturity estimates were determined considering the body length, weight, and external characteristics. In all examinations, the testes were discerned by the presence of a hyperechoic central line, called the mediastinum testis, a landmark for their identification during ultrasonography. No significant differences in echogenicity were detected on the ultrasonographic appearance of the testes among the studied animals. On adult male botos, apparent parenchymal nodulation of the testis was observed on scanning in most of the animals and probably constituted evidence of reproductive maturity. Using the color Doppler technique, blood flow was detected along the mediastinum testis that progressively decreased toward the periphery of this organ. Little blood flow could be identified by color Doppler. Power Doppler allowed better accuracy to identify testicular vessels, their topography, and their differentiation from adjacent structures. Ultrasonographic examination provides useful data for morphologic characterization of the boto's testes. Examination using Doppler techniques was considered a valuable tool to evidence blood flow through the testicular parenchyma.

Noninvasive monitoring of androgens in male Amazonian manatee (Trichechus inunguis): biologic validation.

The Amazonian manatee (Trichechus inunguis) is endemic in the Amazonian basin and is the only exclusively fresh water sirenian. Historically hunted on a large scale, this species is now considered endangered, and studies on the reproductive physiology are critical for the improvement of reproductive management of captive and wild populations of manatees. The aim of this study was to verify the viability of androgen measurement in saliva, lacrimal, urine, and fecal samples of the Amazonian manatee by conducting a hormone challenge. Two adult male manatees (A-1 and A-2) were submitted to an experimentation protocol of 12 day (D1 to D10). On D0, the animals received an intramuscular injection of gonadotropin-releasing hormone (GnRH)-analogue. Salivary, lacrimal, urinary, and fecal samples were collected daily (between 0800 hours and 0900 hours) and frozen at -20 degrees C until assayed. Fecal samples were lyophilized, extracted with 80% methanol, and diluted in buffer before the radioimmunoassay (RIA). Urine samples underwent acid hydrolysis and were diluted in depleted bovine serum. Salivary and lacrimal samples were assayed without the extraction step. Hormonal assays were conducted with a commercial testosterone RIA kit. An androgen peak (> median + 2 interquartile range [IQR]) was observed in all matrices of both animals, although it was less prominent in the lacrimal samples of A-2. However, the fecal androgen peak (A-1 peak = 293.78 ng/g dry feces, median [IQR] = 143.58 [32.38] ng/g dry feces; A-2 peak = 686.72 ng/g dry feces, median [IQR] = 243.82 [193.16] ng/g dry feces) occurred later than urinary (A-1 peak = 648.16 ng/mg creatinine [Cr], median [IQR] = 23.88 [30.44] ng/mg Cr; A-2 peak = 370.44 ng/mg Cr, median [IQR] = 113.87 [117.73] ng/mg Cr) and salivary (A-1 peak = 678.89 pg/ml, median [IQR] = 103.69 [119.86] pg/ml; A-2 peak = 733.71 pg/ml, median [IQR] = 262.92 [211.44] pg/ml) androgen peaks. These intervals appear to be correlated with the long digesta passage time in this species. The salivary and urinary peaks were closely associated. These results demonstrate that androgen concentrations in saliva, urine, or feces samples reflect reliably physiologic events and are a powerful tool for noninvasive reproductive monitoring of Amazonian manatees.

Reproductive anatomy of the female Amazonian manatee Trichechus inunguis Natterer, 1883 (Mammalia: Sirenia).

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.

Phylogeography and population genetics of the endangered Amazonian manatee, Trichechus inunguis Natterer, 1883 (Mammalia, Sirenia).

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.