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.

Ancestral chromosomal signatures of Paenungulata (Afroteria) reveal the karyotype of Amazonian manatee (Trichechus inunguis, Sirenia: Trichechidae) as the oldest among American manatees.

BACKGROUND: Chromosomal painting in manatees has clarified questions about the rapid evolution of sirenians within the Paenungulata clade. Further cytogenetic studies in Afrotherian species may provide information about their evolutionary dynamics, revealing important insights into the ancestral karyotype in the clade representatives. The karyotype of Trichechus inunguis (TIN, Amazonian manatee) was investigated by chromosome painting, using probes from Trichechus manatus latirostris (TML, Florida manatee) to analyze the homeologies between these sirenians. RESULTS: A high similarity was found between these species, with 31 homologous segments in TIN, nineteen of which are whole autosomes, besides the X and Y sex chromosomes. Four chromosomes from TML (4, 6, 8, and 9) resulted in two hybridization signals, totaling eight acrocentrics in the TIN karyotype. This study confirmed in TIN the chromosomal associations of Homo sapiens (HSA) shared in Afrotheria, such as the 5/21 synteny, and in the Paenungulata clade with the syntenies HSA 2/3, 8/22, and 18/19, in addition to the absence of HSA 4/8 common in eutherian ancestral karyotype (EAK). CONCLUSIONS: TIN shares more conserved chromosomal signals with the Paenungulata Ancestral Karyotype (APK, 2n = 58) than Procavia capensis (Hyracoidea), Loxodonta africana (Proboscidea) and TML (Sirenia), where TML presents less conserved signals with APK, demonstrating that its karyotype is the most derived among the representatives of Paenungulata. The chromosomal changes that evolved from APK to the T. manatus and T. inunguis karyotypes (7 and 4 changes, respectively) are more substantial within the Trichechus genus compared to other paenungulates. Among these species, T. inunguis presents conserved traits of APK in the American manatee genus. Consequently, the karyotype of T. manatus is more derived than that of T. inunguis.

Assessment of lipids and adrenal hormones in the Florida manatee (Trichechus manatus latirostris) from different habitats.

Florida manatees (Trichechus manatus latirostris), a federally protected species, are classified as threatened due to anthropogenic stressors. Manatees inhabit sites that are impacted by human activities that can negatively affect stress physiology and metabolism. Samples collected from healthy manatees (pregnant females, non-pregnant females, and males) at Crystal River and Indian River Lagoon in Florida, were assessed for adrenal hormones, proteins, glucose, and lipid content in plasma. The objective was to determine if healthy manatees sampled between 2010-2014 from the Indian River Lagoon exhibited evidence of stress compared to healthy manatees sampled between 2012-2019 from Crystal River. Plasma cortisol concentrations were not different in male and non-pregnant female manatees between sites but were elevated in pregnant manatees. Plasma aldosterone concentrations were elevated in Indian River Lagoon manatees relative to those at Crystal River, possibly due to differences in salinity and available freshwater between the two environments. Site differences were noted for plasma protein and glucose concentrations in manatees; additionally, differences between the sexes were also observed in glucose concentrations. Fifteen lipid subclasses, including oxidized lysophosphatidylcholines, oxidized phosphatidylcholines, oxidized triacylglycerols, were elevated in manatees from the Indian River Lagoon relative to manatees from Crystal River. Evidence of a stress response in healthy Indian River Lagoon manatees was lacking compared to Crystal River manatees. Differences in metabolites related to energy (glucose, protein, and lipids) may be related to site-specific variables, such as salinity and food availability/quality. This study generates novel data on plasma lipid profiles and provides cortisol, aldosterone, glucose, and protein values from healthy Florida manatees in two disparate sites that can be referenced in future studies. These data contribute to an improved understanding of manatee physiology to better inform population management.

Environmental characterization of home range of Antillean manatees (Trichechus manatus) released in northeastern Brazil.

The Antillean manatee occurs discontinuously from the state of Amapá to the state of Alagoas on the coast of Brazil. There is also evidence of reintroduced manatees using the coasts of Sergipe and Bahia, with a preference for calm shallow waters. This study characterized the home range areas of six rehabilitated manatees released in northeastern Brazil. The activities were conducted in the states of Paraíba, Sergipe, and Bahia. Type of environment, substrate, depth, aquatic vegetation, physicochemical variables of the water, presence of solid waste, human settlements, and watercraft were considered to characterize the areas. The results showed a manatee preference for sheltered areas. Resources were available in larger quantities in the dry season, and a reduction in the availability of food items was fund over the years. High overlap was found in the multivariate space of the individuals in terms of the characteristics of the habitats. The estuary of the Paraíba River and the coastal area of Cabedelo Beach in Paraíba showed the greatest amount of solid waste, human settlements, and watercraft. Released manatees exhibited a preference for sites shallower than two meters, with food resources and fresh water availability.

First molecular identification of the trematode Pulmonicola cochleotrema (Platyhelminthes: Opisthotrematidae) in West Indian manatees (Trichechus manatus, Sirenia: Trichechidae) from Puerto Rico and Florida.

West Indian manatees (Trichechus manatus) harbor a variety of endoparasites, including the nasal trematode Pulmonicola cochleotrema, which infects the respiratory tract, especially the nasal passages. Previous studies have described and identified this digenean using morphological data only. This study presents the first molecular identification of P. cochleotrema in West Indian manatees from Puerto Rico and Florida. Samples of the trematode were collected from seven manatees found stranded dead at both locations. The small subunit ribosomal DNA (18S rDNA) was amplified from each sample using universal primers for different regions of the gene, resulting in a consensus sequence of 1871 base pairs. The phylogenetic reconstruction was carried out using DNA sequences of other species of digenean parasites from other hosts, including a trematode of the same taxonomic family from another sirenian species. Specimens collected from both locations show the same molecular identity using SSU rDNA sequence data. The identity of P. cochleotrema was confirmed using the Basic Local Alignment Search Tool from the National Center for Biotechnology Information database, yielding a high similarity of 98.8 % with Opisthotrema dujonis and 98.2 % with Lankatrema mannarense located in the same clade in our analysis. The latter two digeneans belong to the Opisthotrematidae as does P. cochleotrema and previous studies reported them infecting the Eustachian tubes, esophagus, and digestive tract in dugongs (Dugong dugon). These findings evidence that the nasal trematode of manatees in Florida and Puerto Rico and the dugong, all inhabiting and feeding in marine environments, will have a marine mollusk as an intermediate host, probably a gastropod. The question remains, which species of nasal trematode are harbored by lotic-dwelling manatees in other parts of their distribution like South America.

PRELIMINARY EVALUATION OF SYMMETRIC DIMETHYLARGININE AS A BIOMARKER OF RENAL INSUFFICIENCY IN WILD AND REHABILITATED FLORIDA MANATEES (TRICHECHUS MANATUS LATIROSTRIS).

Currently, there are limited antemortem tests available to evaluate renal function in Florida manatees (Trichechus manatus latirostris). Although few reports of renal pathology in manatees exist in the veterinary literature, debilitated animals that present to rehabilitation centers are frequently dehydrated, may have sustained renal trauma secondary to watercraft injury, and may experience ischemic events related to coagulation disorders leading to renal compromise. Clinicians remain limited to the analysis of blood urea nitrogen, creatinine levels, and urinalysis (if urine is obtained) to determine the extent of renal insufficiency, which may not accurately reflect renal function. This poses a diagnostic challenge for clinicians in discerning how critical renal compromise is to the animal's overall health and prognosis. For the first phase of this study, retrospective symmetric dimethylarginine (SDMA) values were determined from banked serum or plasma samples from 14 wild Florida manatees that had been collected while the animals were under rehabilitation in zoological institutions prior to their deaths. Values of SDMA for nine samples from eight manatees with known renal disease, as determined by histopathology, were compared to SDMA values for seven samples from six manatees with no reported renal lesions on histopathology. Values of SDMA from wild Florida manatees with known renal disease (mean 33.56 µg/dl ± 13.15, P= 0.017) were statistically significantly elevated when compared to SDMA values from manatees with no reported renal lesions (mean = 18.71 µg/dl ± 6.9) on histopathology. For the second phase of the study, serum or plasma samples were obtained from wild manatee populations from two geographically separate presumed-healthy wild manatee populations (n = 57). Although the upper limit was higher, serum SDMA concentrations from presumed-healthy wild manatees were comparable to those reported in small animal and equine medicine at 5.88-16.97 µg/dl.

Differential MC5R loss in whales and manatees reveals convergent evolution to the marine environment.

Melanocortin 5 receptor (MC5R), which is expressed in the terminally differentiated sebaceous gland, is a G protein-coupled receptor (GPCR). MC5R exists mostly in mammals but is completely lost in whales; only the relic of MC5R can be detected in manatees, and phenotypically, they have lost sebaceous glands. Interestingly, whales and manatees are both aquatic mammals but have no immediate common ancestors. The loss of MC5R and sebaceous glands in whales and manatees is likely to be a result of convergent evolution. Here, we find that MC5R in whales and manatees are lost by two different mechanisms. Homologous recombination of MC5R in manatees and the insertion of reverse transcriptase in whales lead to the gene loss, respectively. On one hand, in manatees, there are two "TTATC" sequences flanking MC5R, and homologous recombination of the segments between the two "TTATC" sequences resulted in the partial loss of the sequence of MC5R. On the other hand, in whales, reverse transcriptase inserts between MC2R and RNMT on the chromosome led to the loss of MC5R. Based on these two different mechanisms for gene loss in whales and manatees, we finally concluded that MC5R loss might be the result of convergent evolution to the marine environment, and we explored the impact on biological function that is significant to environmental adaptation.

The first assessment of the genetic diversity and structure of the endangered West Indian manatee in Cuba.

The coastal waters of Cuba are home to a small, endangered population of West Indian manatee, which would benefit from a comprehensive characterization of the population's genetic variation. We conducted the first genetic assessment of Cuban manatees to determine the extent of the population's genetic structure and characterize the neutral genetic diversity among regions within the archipelago. We genotyped 49 manatees at 18 microsatellite loci, a subset of 27 samples on 1703 single nucleotide polymorphisms (SNPs), and sequenced 59 manatees at the mitochondrial control region. The Cuba manatee population had low nuclear (microsatellites HE = 0.44, and SNP HE = 0.29) and mitochondrial genetic diversity (h = 0.068 and π = 0.00025), and displayed moderate departures from random mating (microsatellite FIS = 0.12, SNP FIS = 0.10). Our results suggest that the western portion of the archipelago undergoes periodic exchange of alleles based on the evidence of shared ancestry and low but significant differentiation. The southeast Guantanamo Bay region and the western portion of the archipelago were more differentiated than southwest and northwest manatees. The genetic distinctiveness observed in the southeast supports its recognition as a demographically independent unit for natural resource management regardless of whether it is due to historical isolation or isolation by distance. Estimates of the regional effective population sizes, with the microsatellite and SNP datasets, were small (all Ne < 60). Subsequent analyses using additional samples could better examine how the observed structure is masking simple isolation by distance patterns or whether ecological or biogeographic forces shape genetic patterns.

Manatee cognition in the wild: an exploration of the manatee mind and behavior through neuroanatomy, psychophysics, and field observations.

Cognition refers to the mechanisms for acquiring, processing, storing, and acting on information, all of which are critical to understanding the behavior of animals. These mechanisms are poorly known in manatees, especially how they are expressed in the wild. To expand our understanding of manatee cognition, we gathered information from behavioral experimentation in the laboratory, neuroanatomical research, controlled field studies, integrated laboratory and field measurement, and natural history observations (published reports, written surveys, and interviews with knowledgeable observers). Laboratory research, both neuroanatomical and behavioral, provided the most empirical data, primarily on sensory/perceptual capacities. Inferences from these data and narratives from surveys and interviews illuminated possibilities for higher order cognition. Evidence from field measurements was sparse, although substantial amounts of information have been collected from tracking data and to a lesser extent vessel impact studies, which can be used to infer cognitive attributes. Manatees are tactile-auditory specialists with complementary visual and chemosensory abilities. They demonstrate learning characteristics typical of vertebrates. Movement tracking data plus direct observations suggest that they have good spatial cognition, indicated by their ability to traverse complicated water networks and memory for foraging and warm water sites. They engage in a wide range of play-like, object manipulation, and mimetic behaviors, which suggests cognitive capacities beyond basic associative learning. Understanding manatee cognition beyond the laboratory will be necessary for conservation of manatees as they face challenges such as habitat degradation and threats from water-borne vessel traffic. There is a clear need for more direct research in natural settings.

Cognition of the manatee: past research and future developments.

In this paper, we present a review of the current knowledge related to the cognitive abilities of the manatee, with a focus on the Antillean manatee in situ and ex situ. Following a biocentric approach, we consider the animals' ecology, perception and sociality and we introduce future perspectives on their cognition. Scientific literature on the cognitive abilities of Antillean manatees' is limited and mainly linked to medical training and veterinary manipulations. To perceive and to interact with their social and natural environment (e.g. social interactions, foraging and traveling), manatees use visual, acoustic and tactile modalities that may be involved in a large range of cognitive abilities. Research on stimuli perception in manatees is scarce; however, these animals demonstrate abilities to learn and appear to show long-term memory. For example, to mate and/or to forage manatees travel at medium and large geographical scales; without doubt their movements entail the use of a set of stimuli and learning processes. Furthermore, their social skills (e.g. social organization, tactile and acoustic communications) are also poorly understood although their social interactions appear to be more complex than previously thought. Finally, as for many animals, temperament/personality may play a key role during their interactions with conspecifics and the environment. These aspects on manatee behavior and cognition are important for management and conservation purposes and help us understand the evolution of these marine mammals.

Manatee Trichechus manatus latirostris calf mortality in Florida: a retrospective review of pathology data from 2009-2017.

High maternal investment and extended inter-calving intervals in Florida manatees Trichechus manatus latirostris make calf survivorship critical to overall population growth. However, detailed patterns of causes of mortality in calves have not been reported and state agency statistics report portions of perinatal mortality based on body length rather than actual cause of death (COD). The objectives of this study were to categorize COD based on necropsy data and geographical location in Florida for 1209 manatee calf carcasses (<236 cm total length) examined between January 2009 and December 2017 and to describe factors contributing to calf mortality. Results indicated COD was attributed to natural causes (47%, n = 573), cold stress syndrome (38%, n = 457), watercraft injury (13%, n = 155), or other human-related causes (2%, n = 24). Natural causes were the leading COD for small calves <151 cm, with death due to stillbirth or dystocia most frequent (48%, n = 273/573). Enteric trematodiasis contributed to a large proportion of deaths from natural causes in large calves within the southwest region of Florida, with an increasing annual trend. Brevetoxicosis contributed substantially to natural causes within the southwest region exclusively and was commonly comorbid with enteric trematodiasis. Cold stress syndrome was the leading cause of death for large calves (151-235 cm), with the Atlantic region having the highest proportion of cases. Watercraft injury was a sustained threat to large calves, especially within the southwest region. This report provides details on specific health threats and patterns of mortality among manatee calves.

First characterization of vocalizations and passive acoustic monitoring of the vulnerable African manatee (Trichechus senegalensis).

Even among the understudied sirenians, African manatees (Trichechus senegalensis) are a poorly understood, elusive, and vulnerable species that is difficult to detect. We used passive acoustic monitoring in the first effort to acoustically detect African manatees and provide the first characterization of their vocalizations. Within two 3-day periods at Lake Ossa, Cameroon, at least 3367 individual African manatee vocalizations were detected such that most vocalizations were detected in the middle of the night and at dusk. Call characteristics such as fundamental frequency, duration, harmonics, subharmonics, and emphasized band were characterized for 289 high-quality tonal vocalizations with a minimum signal-to-noise ratio of 4.5 dB. African manatee vocalizations have a fundamental frequency of 4.65 ± 0.700 kHz (mean ± SD), duration of 0.181 ± 0.069 s, 97% contained harmonics, 21% contained subharmonics, and 27% had an emphasized band other than the fundamental frequency. Altogether, the structure of African manatee vocalizations is similar to other manatee species. We suggest utilizing passive acoustic monitoring to fill in the gaps in understanding the distribution and biology of African manatees.

Brucella Infection Investigation in Cetaceans and Manatees in Northeast Brazil.

Among the bacterial infections that impair the health status of marine mammals, those caused by Brucella spp. are the most reported worldwide. Brucella infections in marine mammals can result in acute or chronic disease and are associated with variable clinical outcomes, depending on the organ involved during the infectious process, infection route, host immunity, and strain pathogenicity. Asymptomatic infections may also occur. The current study expands the investigation of Brucella infection in northeast Brazil by analyzing 19 dead, stranded cetaceans and 52 Antillean manatees Trichechus manatus manatus. The manatees included 8 dead, captive manatees and 44 live specimens, of which 10 were analyzed only after reintroduction into the wild as part of a rehabilitation program, 9 were analyzed both while in captivity or semi-captivity and after reintroduction, 20 were sampled only in captivity or semi-captivity, and 5 were free-living manatees. Serological tests were used to screen for antibodies against smooth Brucella spp. Whole blood, swabs, and tissue samples were screened for Brucella spp. DNA by PCR. Samples with positive PCR results were cultured for Brucella spp. isolation. All manatees yielded negative results in serological and molecular tests. Brucella spp. DNA was detected in the kidney of one adult Guiana dolphin Sotalia guianensis exhibiting necrosis in the liver. No growth of Brucella spp. was observed via microbiological culturing. This study is the first report of Brucella spp. DNA detection in cetaceans in the state of Pernambuco, and it highlights the importance of conducting systematic monitoring for the presence of Brucella infection in marine mammals along the Brazilian coast, especially in the northeast region, where several cases have been reported.

PRESENTATION AND MANAGEMENT OF DENTAL PAD FRACTURES IN TWO FLORIDA MANATEES (TRICHECHUS MANATUS LATIROSTRIS).

The Florida manatee (Trichechus manatus latirostris) has well-developed keratinized dental pads at the most rostral aspect of their mouth to assist with mastication. This unique development is thought to be an adaptive response to their highly abrasive diets that contain phytoliths and sediments that may accelerate dental wear. In May 2013, two Florida manatees presented with multiple fractures in their inferior dental pads. The fractures were successfully managed with nutritional modifications, dental pad trimming, and vigilant monitoring through behavioral husbandry training. Signs of spontaneous healing were observed as early as 60 days after initial presentation with subsequent full resolution. Although surgical intervention was planned, the spontaneous healing mitigated significant health risks associated with the procedure. To the authors' knowledge, these are the first reported cases of dental pad fractures and their spontaneous healing and resolution in manatees.

EVALUATION OF THE PHARMACOKINETIC BEHAVIOR OF TULATHROMYCIN (DRAXXIN) IN FLORIDA MANATEES (TRICHECHUS MANATUS LATIROSTRIS) UNDERGOING MEDICAL REHABILITATION.

Florida manatees (Trichechus manatus latirostris) frequently present to rehabilitation care facilities for various conditions, including boat strike trauma, cold stress syndrome, and brevetoxicosis. Throughout the course of treatment, antimicrobial use to treat respiratory disease is frequently warranted. To date, clinicians have extrapolated dosages based on established information available in bovine and equine medicine. The routes of administration, efficacy, and treatment intervals are considerations in dealing with critical wild animals. The use of tulathromycin, a triamilide antibiotic, has been studied in multiple domestic species of economic importance, including cattle, small ruminants, and swine, and has revealed efficacy against respiratory diseases. Given this information, this antibiotic has also been used in manatees with positive clinical outcomes. This study employed sparse sampling and evaluated banked plasma samples at various time intervals post-tulathromycin administration obtained during the clinical treatment course of nine animals during their rehabilitation. Preliminary pharmacokinetic analysis following administration of a single dose estimated a half-life of 33.75 h and volume of distribution per fraction absorbed (Vz/F = 4.29 L/kg). The pharmacokinetic behavior of tulathromycin in Florida manatees can be used to optimize dosage regimens in this species.

For Better, For Worse: Manatee-Associated Tursiocola (Bacillariophyta) Remain Faithful to Their Host.

With the advent of more comprehensive research into the microbiome and interactions between animals and their microbiota, new solutions can be applied to address conservation challenges such as husbandry and medical care of captive animals. Although studies on epizoic algae are relatively rare, and the function and role of those mainly photosynthetic organisms in the animal microbiome is not well understood, recent surveys on epizoic diatoms show that some of them exhibit traits of obligate epibionts. This study explores diatom communities on captive-born manatees from the Africarium in Wroclaw, Poland. Light and scanning electron microscopy analyses revealed that skin of all animals sampled was dominated by apochlorotic Tursiocola cf. ziemanii, an epizoic species described recently from Florida manatees, that reached 99,9% of the total diatom abundance. Despite using media with a wide range of salinity (0-34), the isolated Tursiocola cells did not grow, whereas the normally pigmented Planothidium sp., that was only occasionally found on the animal substratum, survived in all culture media tested. Our observations provide direct evidence that manatee-associated Tursiocola endure the dramatic salinity changes that occur regularly during their host life cycle, and can thrive in an artificial captive setting, if the manatee substratum is available. The impact of practices and routines used by the Africarium on manatee-associated diatoms, as well as ultrastructure of areolae in Tursiocola, are briefly discussed.

Independent erosion of conserved transcription factor binding sites points to shared hindlimb, vision and external testes loss in different mammals.

Genetic variation in cis-regulatory elements is thought to be a major driving force in morphological and physiological changes. However, identifying transcription factor binding events that code for complex traits remains a challenge, motivating novel means of detecting putatively important binding events. Using a curated set of 1154 high-quality transcription factor motifs, we demonstrate that independently eroded binding sites are enriched for independently lost traits in three distinct pairs of placental mammals. We show that these independently eroded events pinpoint the loss of hindlimbs in dolphin and manatee, degradation of vision in naked mole-rat and star-nosed mole, and the loss of external testes in white rhinoceros and Weddell seal. We additionally show that our method may also be utilized with more than two species. Our study exhibits a novel methodology to detect cis-regulatory mutations which help explain a portion of the molecular mechanism underlying complex trait formation and loss.

Survey of Brucella spp. and Leptospira spp. antibodies in cetaceans and manatees of the Amazon basin and Atlantic Ocean, Brazil.

Aquatic mammals can act as sentinels of emerging and resurging pathogens in the environment. Brucella spp. and Leptospira spp. are 2 zoonotic pathogens relevant to aquatic mammals, and their detection can be used to assess pathogen exposure. In this study, serum from 84 individuals-63 cetaceans (families Iniidae, n = 37; Delphinidae, n = 22; and Kogiidae, n = 4) and 21 West Indian manatees Trichechus manatus-was tested by the Rose Bengal Test (RBT) and a commercial competitive enzyme-linked immunosorbent assay (c-ELISA) for detecting Brucella spp. antibodies, and the microscopic agglutination test (MAT) for screening Leptospira spp. exposure. Overall, 4.8% (3/63) of cetaceans were positive by RBT and 15.9% (10/63) by c-ELISA for Brucella spp. Serum from 8 c-ELISA positive cetaceans (with available serum) was further tested via serum agglutination test (SAT) and 1 individual was positive. c-ELISA was more sensitive than RBT. Exposure to Brucella spp. was found in 5 cetacean species: Clymene dolphin Stenella clymene, short-finned pilot whale Globicephala macrorhynchus, pygmy killer whale Feresa attenuata, melon-headed whale Peponocephala electra and Atlantic bottlenose dolphin Tursiops truncatus in the Atlantic Ocean, Brazil, expanding the range of known Brucella seropositive aquatic hosts. No evidence of Brucella spp. exposure was found in Iniidae and Kogiidae odontocetes and manatees. Antibodies against Leptospira spp. were not detected in cetaceans and sirenians by MAT. These results contribute to the evaluation of different Brucella spp. serological methods in cetaceans and manatees and highlight the epidemiology of zoonotic pathogens in aquatic mammals of the southwestern Atlantic Ocean and the Amazon basin.

Cis-Regulatory Evolution of Forkhead Box O1 (FOXO1), a Terminal Selector Gene for Decidual Stromal Cell Identity.

Studies in human and mouse have shown that decidual stromal cells (DSC), which develop in the innermost lining of uterus, mediate placentation by regulating maternal immune response against the fetus and the extent of fetal invasion. Investigating when and how DSC evolved is thus a key step to reconstructing the evolutionary history of mammalian pregnancy. We present molecular evidence placing the origin of DSC in the stem lineage of eutherians (extant placental mammals). The transcription factor forkhead box O1 (FOXO1) is a part of the core regulatory transcription factor complex (CoRC) that establishes the cell type identity of DSC. Decidualization, the process through which DSC differentiate from endometrial stromal fibroblasts, requires transcriptional upregulation of FOXO1 Contrary to other examples in mammals where gene recruitment is caused by the origin of an alternative promoter, FOXO1 is transcribed from the same promoter in DSC as in endometrial stromal fibroblasts. Comparing the activities of FOXO1 promoters from human, mouse, manatee (Afrotheria), and opossum (marsupial) revealed that FOXO1 promoter evolved responsiveness to decidualization signals in the stem lineage of eutherians. This eutherian vs. marsupial pattern of promoter activity was not observed in some other cell types expressing FOXO1, suggesting that this cis-regulatory evolution occurred specifically in the context of the origin of DSC. Sequence comparison revealed eutherian-specifically conserved nucleotides that contribute to the eutherian promoter activity. We conclude that the cis-regulatory activity of a terminal selector gene for decidual stromal cell identity evolved in the stem lineage of eutherians supporting a model where decidual cells are a eutherian innovation.