ABSTRACT Although there are several studies on the skull of Amazonian manatees (Trichechus inunguis) to better understand the anatomy, physiology, and behavior of these animals, the analysis of the brain has been often neglected. Typically, in osteological studies, the brain is discarded to preserve the integrity of the skull. One of the main reasons for this neglect of the brain is the lack of adequate dissection protocols to allow the extraction of the intact brain while preserving the integrity of the skull. In this study, we present a simple step-by-step protocol for a comprehensive procedure of brain extraction and fixation in manatee calves while ensuring the preservation of the skull structure to the best possible extent for further studies. The protocol is based on an incision at the posterior part of the skull, extending laterally toward the parietal bone until reaching the frontal bone, followed by removing the upper portion of the skullcap to extract the brain. After the procedure, the removed skull portion can be reconstituted to preserve the entire skull structure. The protocol also offers adaptations to simplify the methodology according to the reality of places with little laboratory structure, allowing the preservation of rare tissues with limited resources and/or in areas of difficult access. Our proposed methodology enables maximum utilization of the collected animal, which not only aligns with ethical and practical considerations, but also makes material available for a detailed description of the manatee brain, and a better understanding of the neuroanatomy of aquatic mammals in general.

Abstract Animals adopt various behavioral strategies to meet their biological needs, often adjusting their activity cycles. While some species restrict their activities to specific periods within the 24-hour light and dark cycle, others are cathemeral, showing flexible activity patterns that include both day and night. This study investigates the cathemeral activity of Amazonian manatees (Trichechus inunguis) in Anavilhanas National Park, Brazil, with a focus on their nocturnal behavior and ecological adaptability. Using thermal cameras, we recorded nocturnal feeding for the first time, highlighting the manatees’ flexibility beyond the typical diurnal–nocturnal cycle. Our findings reveal that manatees adjust their feeding strategies according to seasonal vegetation availability and water levels. Specifically, they feed at night on the riparian plant maracarãna (Coccoloba densifrons), which is only accessible during the flood season. This nocturnal behavior likely helps minimize predation risk and enhances foraging efficiency. These insights significantly improve our understanding of manatee ecological behavior in the Amazon, demonstrating their adaptability to environmental changes. The study underscores the importance of considering cathemeral activity in conservation strategies to ensure the ongoing protection of Amazonian manatees against environmental and human pressures.

Evaluating renal function is essential for managing captive wild animals, particularly threatened species like the Amazonian manatee (Trichechus inunguis) in rehabilitation and prerelease programs. A series of urine diagnostic tests, such as gross appearance, semiquantitative chemical analyses, microscopic review of sediments, and quantitative analyses of urea and creatinine, were performed in 57 free-catch urine samples. On the same occasion, 52 serum samples from the same individuals were analyzed for creatine kinase activity, creatinine, blood urea nitrogen, albumin, sodium, potassium, and chloride concentrations; serum symmetric dimethylarginine (SDMA) was measured for the first time in the species. Females had higher urine creatinine concentration (21.7 ± 11.8 mg/dl) and specific gravity (1.003 ± 0.003 mg/dl) than males (11.9 ± 8.5 mg/dl and 1.001 ± 0.002 mg/dl) (P < 0.01 for both). Males presented higher urine creatinine:urea ratio than females (24.8 ± 29.2 and 20.5 ± 29.2, respectively) (P < 0.05). Urine pH was lower in manatees from the semicaptive area (7.8 ± 0.6) than in manatees under human care (8.5 ± 0.6) (P = 0.02) using the benchtop pH meter but showed no difference between areas in the dipstick reaction (P = 0.71), given its lower sensitivity and narrower range detection. Furthermore, 18 and 75% of urine samples were positive for the presence of blood and/or nitrite, respectively, in the dipstick reaction, possibly because of sample contamination. Significant correlation was observed between serum blood urea nitrogen and urine urea concentrations (P = 0.02), and between serum and urine creatinine concentrations (P = 0.01). Serum SDMA did not differ between sex or between captive and semicaptive individuals. However, it positively correlated with urine (P = 0.02) and serum creatinine (P = 0.01) demonstrating its potential as a bioindicator of kidney function. The renal function of Amazonian manatees can be effectively assessed by urinalysis, especially when combined with serum biochemistry.

Abstract For many species at risk, monitoring challenges related to low visual detectability and elusive behavior limit the use of traditional visual surveys to collect critical information, hindering the development of sound conservation strategies. Passive acoustics can cost‐effectively acquire terrestrial and underwater long‐term data. However, to extract valuable information from large datasets, automatic methods need to be developed, tested and applied. Combining passive acoustics with deep learning models, we developed a method to monitor the secretive Amazonian manatee over two consecutive flooded seasons in the Brazilian Amazon floodplains. Subsequently, we investigated the vocal behavior parameters based on vocalization frequencies and temporal characteristics in the context of habitat use. A Convolutional Neural Network model successfully detected Amazonian manatee vocalizations with a 0.98 average precision on training data. Similar classification performance in terms of precision (range: 0.83–1.00) and recall (range: 0.97–1.00) was achieved for each year. Using this model, we evaluated manatee acoustic presence over a total of 226 days comprising recording periods in 2021 and 2022. Manatee vocalizations were consistently detected during both years, reaching 94% daily temporal occurrence in 2021, and up to 11 h a day with detections during peak presence. Manatee calls were characterized by a high emphasized frequency and high repetition rate, being mostly produced in rapid sequences. This vocal behavior strongly indicates an exchange between females and their calves. Combining passive acoustic monitoring with deep learning models, and extending temporal monitoring and increasing species detectability, we demonstrated that the approach can be used to identify manatee core habitats according to seasonality. The combined method represents a reliable, cost‐effective, scalable ecological monitoring technique that can be integrated into long‐term, standardized survey protocols of aquatic species. It can considerably benefit the monitoring of inaccessible regions, such as the Amazonian freshwater systems, which are facing immediate threats from increased hydropower construction.

Manatee populations are declining worldwide, and all currently existing species are considered vulnerable by the IUCN. The most common problems during nurturing young Amazonian manatees, Trichechus inunguis, in rescue centres are of gastrointestinal nature leading to inappetence, diarrhoea, cachexia and even death. Endoparasites play an important role in the well-being of wildlife in captivity as well as in the wild, though information about relevant protozoan and metazoan endoparasites in Amazonian manatees is still scarce. Therefore, this study aimed to find endoparasites in T. inunguis by analyzing faecal samples from 23 Amazonian manatees which were kept in rescue centres in the Peruvian Amazon. The samples were screened for protozoan and metazoan parasites using coproscopical analysis and molecular tools. Out of twenty juvenile animals eleven were positive for at least one Eimeriidae. Two morphologically different, not yet genetically described Eimeria species were identified. One of them seems to be Eimeria trichechi which has only been described once in 1984 in Amazonian manatees from Brazil. It was not found to lead to clinical symptoms of coccidiosis in this study. The second, Eimeria sp. Type B was associated with clinical coccidiosis in a young Amazonian manatee, which showed gastrointestinal symptoms including diarrhoea, inappetence and cachexia. No other protozoan or metazoan parasite were detected in any of the samples. The present study is the first to investigate endoparasites in Amazonian manatees using molecular tools and is the first to identify an Eimeria species that could be associated with clinical symptoms in T. inunguis. With information from our study rescue centres can improve monitoring of parasites more effectively to reduce morbidity and mortality rates among rehabilitated manatees as well as improve the health status and fitness of animals for a successful release back into the wild.

A giant gardener of the Floodplains: The Amazonian manatee

ABSTRACT: The Amazonian manatee (Trichechusinunguis) is an aquatic mammal threatened with extinction. However, few studies have investigated the pathogens in this species, which may affect both animal and human health. This study aimed to evaluate the frequency, distribution, and patterns of antimicrobial susceptibility of Staphylococcus spp. and Escherichiacoli colonizing the nasal and rectal cavities of Amazonian manatees kept in captivity at the National Institute for Amazonian Research (INPA) in the state of Amazonas, Brazil. Rectal and nasal swabs from 44 manatees of different ages were used in this study. The genus Staphylococcus was isolated from the nasal swabs of 32 (72.7%) animals, with two individuals harboring more than one species of Staphylococcus. S. sciuri was the most commonly isolated species. Resistance to penicillin was observed in 13 (40.6%) isolates, more frequent than the other antimicrobials tested (P = 0.01). E. coli was isolated from the rectal swabs of all animals, with phylogroup B1 being the most frequent among the strains obtained (P = 0.0008). Four isolates (6.8%) were positive for virulence factors, three of which were classified as enterotoxigenicE. coli (ETEC) and one as enteropathogenicE. coli (EPEC). To our knowledge, this is the first study to evaluate Staphylococcus spp. and E. coli in Amazonian manatee samples. This study revealed nasal colonization by Staphylococcus spp., mainly S. sciuri, and diarrheagenicE. coli isolates, including antimicrobial-resistant strains.

Detection of many threatened aquatic mammals, such as manatees (Trichechus spp.), using traditional visual observation methods is associated with high uncertainty due to their low surfacing times, cryptic behaviors, and the environmental heterogeneity of their habitats. Rapid advancements in technology provide an opportunity to address these challenges. In this study, we aimed to quantify survey effort of unoccupied aerial vehicles (UAVs) for detecting the Vulnerable Amazonian manatee (T. inunguis). Using a closed population of manatees that is being rehabilitated within a lake at the Rainforest Awareness, Rescue, and Education Center in Iquitos, Peru, we calculated the number of repeat surveys needed to detect at least one individual with 95% (n = 3.10) and 99% (n = 4.76) confidence. We used both generalized linear mixed-effect models and Bayesian single-species and single-season detection models to determine the effects of the environment (water depth, water transparency, cloud cover, wind speed), time of day, and behavior (breathing, foraging, milling) on the time-to-detection and detection probability, respectively. Both models indicated a significant interaction between water depth and water transparency, causing an increase in the time-to-detection (β = 0.032; 95% CI = 0.028, 0.037) and a decrease in the probability of detecting manatees (α = -0.65; 95% CI = -1.3, -0.007), which was calculated to be 0.62 (95% CI = 0.23, 0.94). Due to the similarities between the lake and in situ habitats, the results of this study could be used to design in situ UAV survey protocols for Amazonian manatees or other difficult-to-detect freshwater aquatic mammals and to monitor ex situ animals pre-and post-release, which should ultimately contribute to a better understanding of their spatial ecology and facilitate data-driven conservation efforts.

Since 1976, Amazonian manatees have been examined either alive or through necropsies as part of a long-term program to rescue and rehabilitate orphan manatee calves whose mothers were either victims of illegal hunting or accidental catch in fishing nets in the Brazilian Amazon region. Rescued manatees have been found to have several types of diseases, but to our knowledge, there is no record of a congenital anomaly of the face, known as an oral cleft, in Amazonian manatees. In this paper, we report the occurrence of an oral cleft in an Amazonian manatee calf, describing the clinical evaluation and discussing the potential causes.

The vulnerable status of the Amazon manatee, Trichechus inunguis, indicates the need to seek measures to guarantee its conservation. In this context, the cultivation of cells in vitro is a strategy that should at least guarantee the preservation of their genetic material. Thus, we established for the first time a primary culture of Amazonian manatee fibroblasts (TINsf) from a skin biopsy of a young male. Karyotypic analysis of the 3rd, 7th, and 12th passages confirmed the taxonomic identity of the species T. inunguis (2n = 56/NF = 92) and indicated that this culture presents genomic stability. Gene and protein expression of vimentin at the 13th passage show the predominant presence of fibroblasts in TINsf. To test the cell line's responsiveness to materials and demonstrate a possible application of this culture, it was exposed to andiroba seed oil (ASO), and its viability and proliferative capacity were evaluated. ASO demonstrated toxic effects at the highest concentrations and longest exposure times tested, reproducing results observed in human cultures, indicating the applicability of TINsf in toxicological and biotechnological studies. After cryopreservation, the TINsf line maintained its proliferative potential, indicating the establishment of a new culture available for future studies.

Progesterone monitoring and gestation length in Amazonian manatees (Trichechus inunguis)

Parasites of Amazonian manatees in a rescue center in Loreto, Peru

The genetic basis underlying adaptive physiological mechanisms has been extensively explored in mammals after colonizing the seas. However, independent lineages of aquatic mammals exhibit complex patterns of secondary colonization in freshwater environments. This change in habitat represents new osmotic challenges, and additional changes in key systems, such as the osmoregulatory system, are expected. Here, we studied the selective regime on coding and regulatory regions of 20 genes related to the osmoregulation system in strict aquatic mammals from independent evolutionary lineages, cetaceans, and sirenians, with representatives in marine and freshwater aquatic environments. We identified positive selection signals in genes encoding the protein vasopressin (AVP) in mammalian lineages with secondary colonization in the fluvial environment and in aquaporins for lineages inhabiting the marine and fluvial environments. A greater number of sites with positive selection signals were found for the dolphin species compared to the Amazonian manatee. Only the AQP5 and AVP genes showed selection signals in more than one independent lineage of these mammals. Furthermore, the vasopressin gene tree indicates greater similarity in river dolphin sequences despite the independence of their lineages based on the species tree. Patterns of distribution and enrichment of Transcription Factors in the promoter regions of target genes were analyzed and appear to be phylogenetically conserved among sister species. We found accelerated evolution signs in genes ACE, AQP1, AQP5, AQP7, AVP, NPP4, and NPR1 for the fluvial mammals. Together, these results allow a greater understanding of the molecular bases of the evolution of genes responsible for osmotic control in aquatic mammals.

Hematology and blood chemistry tests constitute an easy-to-apply veterinary tool that evaluates an organism’s systemic functioning and disease process by comparing the level of specific analytes against species norms. Such analyses help monitor marine mammals’ health and nutritional status. Although reference values have been published for a few manatee populations, there are none for Antillean manatees (Trichechus manatus manatus) in Colombia. We aim to establish the reference values for hematology and serum chemistries for these manatees and determine if there are variations between individuals of different age groups and sex. Thus, we obtained whole blood and serum samples from 45 rehabilitated manatees from Colombia between 1992 and 2021. Complete Blood Count and Comprehensive Metabolic Panel values were calculated, and differences between age groups and sex were determined. Results were compared with published reference intervals of other Antillean manatee populations, Florida manatees (Trichechus manatus latirostris), and Amazonian manatees (Trichechus inunguis) from Brazil. We determined the reference intervals of hematology and serum chemistry for manatees in Colombia for different age and sex categories. No relevant clinical variations were found in hematological parameters due to sex. Marked differences were found between age groups, mainly among young animals with an expected faster metabolism. There were significant variations between hematological and blood chemistry values when the Colombian manatees were compared to manatees from Puerto Rico and Amazonian manatees from Brazil. Such variations are likely influenced by evolutionary history and environmental factors associated with differences in habitat salinity and diet. We recommend that future studies correlate these blood tests with specific panels. We further recommend conducting wild manatee health assessments as this information will yield essential data for species management schemes needed due to the multiple anthropogenic and environmental threats that manatees face today which put the Colombian manatee’s health and ultimate survival at risk.

Cultivated meat (CM) is a disruptive technology that provides an alternative to animal protein. In this context, the Amazon manatee (Trichechus inunguis) emerges as an important case. Although it is illegal to hunt this large mammal, its meat continues to be consumed, causing several threats to its natural habitat. The aim of this study is to explore the impacts of introducing the Amazon manatee CM into the traditional meat value chain as a tool to aid the biodiversity of the Amazon Basin. Thus, we developed a strengths, weaknesses, opportunities, and threats matrix from the content analysis of 11 interviews conducted between October 2021 and May 2022. The interviewees were experts in different fields, ranging from financial analysts of novel food technologies to biologists, researchers, and others. We presented the theme of illegal hunting and its consequences during the interviews, followed by the CM process, and explained how the royalties from the sale of this innovative product could help to preserve Amazon biodiversity through the proposal of a new business model. The main findings suggest that the proposed model would produce good results, but the threat of a rebound effect from the consumption of wild animals was mentioned in most responses, especially by actors involved in conservation. The strengths and opportunities of this disruptive narrative mainly focused on preserving biodiversity and promoting environmental awareness, combining the conservation of wildlife and the consumption of novel food. The weaknesses included the lack of knowledge and the non-existent market. This framework is relevant for policymakers, nongovernmental organizations, and researchers seeking to improve the sustainability not only of the species found in the Amazon, but also around the world.

The last relevant work that reviewed the biological and conservation aspects of the Amazonian manatee (Trichechus inunguis) was published almost 30 years ago. In these three decades, scientific knowledge about the species has advanced substantially. This review article updates knowledge about the various biological and conservation aspects of Amazonian manatee, summarizing the information present in the literature. Topics such as morphology, habitat, distribution, population aspects, food and feedings habits, reproduction, behavior, health, threats, status, and conservation efforts were addressed. The main actions already carried out, as well as the future strategies needed for the conservation of the Amazonian manatee, are also highlighted.

BackgroundChromosomal 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.ResultsA 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).ConclusionsTIN 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.

Morphophysiological species researches are fundamental, and diagnostic imaging is an excellent technique, already used in wild animals, with great application, not invasive and provide real-time information of each body. Amazonian manatees are on the list of endangered animals classified in the vulnerable category and knowledge of the normal pattern of ultrasound anatomy of organs and tissues is important for the maintenance and well-being of captive specimens contributing to reintroduction actions. The objective of the study was to standardize the examination technique and describe the ultrasound findings of the liver, gallbladder, stomach, urinary bladder and the subcutaneous tissue of the abdominal region in Trichechus inunguis, in order to contribute with the anatomical and sonographic knowledge and assist in the diagnosis and prognosis diseases. The study used 18 animals to describe the normal sonographic anatomy in the abdominal cavity of the Amazonian manatee. During abdominal scan, it was possible to visualize the features of the liver, gallbladder, stomach, urinary bladder obtained satisfactory results in this study. Therefore, other structures were not primarily identified by the reduced time, lots of fat and gases in intestines of animals.

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.

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