Verminous bronchitis and pneumonia by nasal trematodes in Greater Caribbean manatees from Puerto Rico.

Five adult Greater Caribbean manatees Trichechus manatus manatus were found stranded on various coasts of Puerto Rico; 2 stranded alive and 3 stranded dead. Clinical signs observed in live-stranded manatees included emaciation, weakness, bradypnea, arrhythmia, and nasal mucus discharge. Postmortem examinations revealed serosanguinous, mucohemorrhagic, or suppurative exudate in bronchi associated with luminal adult Pulmonicola cochleotrema (range: 18-182 trematodes), accompanied by pulmonary abscesses in 2 cases. Histologically, we observed eosinophilic bronchopneumonia of varying severity (n = 4) and chronic erosive to eosinophilic tracheobronchitis (n = 4) with squamous metaplasia (n = 3) and intralesional trematodes and eggs. The trematode identity was confirmed and compared through molecular analysis for the amplified 18S rDNA fragment. Comorbidities included enteric chiorchosis (n = 5), gastric heterocheilosis (n = 4), malnutrition (n = 4), trauma related to watercraft collision (n = 3), systemic toxoplasmosis (n = 1), acute bacterial peritonitis (n = 1), and interstitial nephritis (n = 1), suggesting that immunosuppression was a predisposing factor for lower respiratory tract pulmonicolosis. Based on lesion severity, clinical signs, and the presence and absence of other findings to explain death, this condition was considered the primary cause of death in 1 manatee, a contributory cause of death in 3 manatees, and an incidental finding in 1 individual. These clinicopathological descriptions will facilitate the diagnosis and clinical management of pulmonicolosis in T. manatus, a species endangered with extinction.

Age and sex effects on histological features and in vitro culturing of Antillean manatee (Trichechus manatus manatus, Linnaeus 1758) dermis.

The biobanks from dermal biopsies represent an interesting strategy for biodiversity conservation. Nevertheless, the morphological and cellular patterns of the dermis can be influenced by the age and sex of the individual. Therefore, evaluating these factors is interesting for forming biobanks of Antillean manatees. These animals, representatives of marine fauna, have had their population reduced, and biobanks are essential for their conservation. Then, we evaluated the effects of age (3.5 years vs. 3.6-16 years vs. 23.6 years) and sex (males vs. females) on morphological and cellular parameters using histological and in vitro culture techniques. Regardless of age, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery. Nonetheless, fibroblast reduction was observed in groups aged 23.6 years compared to other animals (p < 0.05). Additionally, cells from animals aged 3.6-16 years showed more significant mitochondrial damage than the other groups (p < 0.05). Regardless of sex, no differences were observed for dermal thickness, collagen fibres, tissue proliferative activity and viable cell recovery; however, females had fewer fibroblasts than males (p < 0.05). Cells from females showed lower mitochondrial damage when compared to cells from males. In summary, although age and sex do not influence dermal thickness and cell recovery, variations in the number of fibroblasts and mitochondrial characteristics were observed among the groups. These differences may be significant for understanding the dermis aspects to be correlated to biobank systems.

Heterocheilus floridensis sp. n. (Nematoda: Heterocheilidae) from the West Indian manatee Trichechus manatus (Trichechidae, Sirenia) in Florida, USA.

Morphological data are used to describe a new nematode species, Heterocheilus floridensis sp. n. (Heterocheilidae) from the digestive tract of the Florida manatee Trichechus manatus latirostris (Harlan) (Trichechidae, Sirenia) from Florida, USA. Examination by light and scanning electron microscopy revealed that the new species differs from the related Heterocheilus tunicatus Diesing, 1839 mainly by having dentigerous ridges on the inner surface of the lips, a median unpaired papilla located anterior to the cloaca, and a considerably larger body size. Sequence data for subunits I and II of mitochondrial cytochrome oxidase gene, 18S small subunit and 28S ribosomal RNA genes were provided for molecular characterisation of the new species. However, the current unavailability of homologous sequence data for congeneric specimens precluded a molecular assessment of the morphological species hypothesis, and ascaridoid phylogenetic hypotheses could not be advanced. Specimens of Heterocheilus sp. collected from the Antillean manatee Trichechus manatus manatus Linnaeus in Puerto Rico, on loan from the US National Museum of Natural History, were morphologically consistent with the new species, so apparently all congeneric nematodes reported from both subspecies of the West Indian manatee Trichechus manatus Linnaeus and previously identified as H. tunicatus belong rather to H. floridensis sp. n. Heterocheilus hagenbecki (Khalil et Vogelsang, 1932) Sprent 1980 is here considered to be a species inquirenda. A key to valid species of Heterocheilus Diesing, 1839 is provided.

Lipidomics and plasma hormone analysis differentiate reproductive and pregnancy statuses in Florida manatees (Trichechus manatus latirostris).

Florida manatees (Trichechus manatus latirostris) are protected as a threatened species, and data are lacking regarding their reproductive physiology. This study aimed to (1) quantify plasma steroid hormones in Florida manatees from two field sites, Crystal River and Indian River Lagoon, at different gestational stages and to (2) identify individual lipids associated with pregnancy status. Ultra-high performance liquid chromatography-tandem mass spectrometric analysis was used to measure plasma steroid hormones and lipids. Pregnant female manatees were morphometrically distinct from male and non-pregnant female manatees, characterized by larger body weight and maximal girth. Progesterone concentrations in manatees were also elevated during early gestation versus late gestation. Cholesterol, an important metabolic lipid, and precursor for reproductive steroids, was not different between groups. Mass spectrometry quantified 949 lipids. Plasma concentrations of glycerophospholipids, glycerolipids, sphingolipids, acylcarnitines, and cholesteryl esters were associated with pregnancy status in the Florida manatee. Most of the lipid species associated with pregnancy were triacylglycerides, phosphatidylethanolamines, and ether-linked phosphatidylethanolamines, which may serve as energy sources for fetal development. This research contributes to improving knowledge of manatee reproductive physiology by providing data on plasma steroid hormones relative to reproductive status and by identifying plasma lipids that may be important for pregnancy. Elucidation of lipid species directly associated with pregnancy has the potential to serve as a diagnostic approach to identify pregnant individuals in fresh and archived samples. These biochemical and morphometric indicators of reproductive status advance the understanding of manatee physiology.

Persistent long-term habitat use by Florida manatees at Fort Pierce, Florida from 1997 to 2020.

To survive cold winters, Florida manatees (Trichechus manatus latirostris) depend on artificial (i.e., power plants) and natural warm water sources such as springs and passive thermal basins. Passive thermal basins can provide critical habitat for manatees for short or extended periods of time. The Henry D. King Powerplant in Fort Pierce, Florida discharged warm water into Moore's Creek until it went offline in 1995. However, it is unknown to what degree manatees continue to occupy this area and how environmental factors influence their occurrence in the creek. To explore this, we examined the habitat use of Florida manatees in Moore's Creek after the shutdown from November 1997 to March 2020 from daily counts of manatees. In addition, we correlated local environmental data (ambient air, temperature, salinity) to assess if Moore's Creek had properties indicative of a passive thermal basin. Results indicated there was not an increase or decrease in habitat use over twenty years in the Creek. The consistent use of Moore's Creek over the study period suggests that this habitat possesses thermal and freshwater resources to support manatee occurrence long-term. These findings provide robust support for the importance of this habitat and passive thermal basins for Florida manatees.

Metal and metalloid maternal transfer in a newborn West Indian manatee (Trichechus manatus) two years after the northeastern oil spill disaster of 2019 in Brazil.

A large-scale oil spill along the northeastern Brazilian coast in 2019-2020 severely impacted primary manatee habitats in Brazil. This study aimed to assess metal and metalloid contamination in a dead manatee calf found following this oil spill. Several elements were detected, including chromium, iron, nickel, lead, and vanadium, which are crude oil components, and thallium, a component of dispersants used to degrade and dissipate crude oil. The presence of these contaminants in the manatee calf is indicative of maternal exposure, metabolism, and newborn transference via placenta and lactation. This is the first report of mother-calf metal transfer in manatees, highlighting the species' vulnerability and potential long-term population dynamics effects, and the first report for several metals and metalloids in these animals, laying the foundation for future research efforts. These findings highlight the urgent need for continued environmental monitoring, ecotoxicological assessments, and conservation initiatives.

Agonistic interactions initiated by adult bottlenose dolphins on Antillean manatee calves in the Caribbean Sea.

The dynamics and drivers of inter-species interactions in the wild are poorly understood, particularly those involving social animal species. Inter-species interactions between cetaceans and sirenians have rarely been documented and investigated. Here, we report 10 cases of interaction initiated by adult bottlenose dolphins (Tursiops truncatus) towards Antillean manatee (Trichechus manatus manatus). Interactions were documented through behavioral observations in the wild (n = 7) and from the examination of orphaned calves (i.e., tooth rake marks on their body; n = 4) that entered a rehabilitation facility, one individual both observed interacting with dolphins and found stranded with bite marks. Bottlenose dolphins were observed interacting with orphan manatee calves and with mother-calf pairs, exhibiting agonistic behavior (n = 2), affiliative or neutral behaviors (n = 1), but the behavioral contexts of these interactions remain unclear in most cases (n = 7). Information on stranded individuals was collected from four calves (of 13 examined calves) recovered in poor condition with bottlenose dolphin tooth rakes and bite wounds on their bodies, one of which died. Injury from bite wounds varied in extent and severity, ranging from superficial scratches leaving rake marks to deep lacerations. Our findings suggest the regular occurrence of agonistic behaviors initiated by adult bottlenose dolphins and directed toward manatee calves. However, the drivers of these interactions remain unknown and need to be further investigated.

Antillean manatee (Trichechus manatus manatus Linnaeus, 1758) Tongue Morphology and Adaptive Herbivorous Implications.

Morphological study of the tongue is an interesting way of understanding evolutionary processes associated with feeding habits. Therefore, the aim of the present study was to describe the tongue morphology of the Antillean manatee and to understand possible morphological relationships with its way of capturing food. Macroscopic dissections and light and scanning electron microscopy analyses of seven manatee tongues were performed. The tongue in Antillean manatees is a muscular and robust organ, divided into apex, body, and root. It is firmly adhered to the floor of the oral cavity. Lingual papillae were distributed over the entire tongue surface. They were identified as filiform papillae concentrated in the apex. Fungiform papillae were present on the apex and lateral regions. Foliate papillae were located on the dorsolateral portion of the root. Lentiform papillae were located across the dorsal tongue surface. The mucosa was lined by a keratinized stratified squamous epithelium presenting compound tubuloacinar glands and taste buds in the foliate papillae. The tongue of the Antillean manatee is similar to other Sirenia species, both of which share a completely herbivorous diet.

Gross and microanatomic description of the dental pads of the Florida manatee, Trichechus manatus latirostris, and their importance to mastication.

OBJECTIVE: Florida manatee feeding ecology is critical to species survival, but the role of dental pads in feeding has received limited attention. This study characterized the gross and microscopic anatomy of the manatee's dorsal and ventral dental pad in relation to these structures' importance in mastication, which furthers our understanding of manatee feeding and health. DESIGN: Whole heads from 6 animals (4 male and 2 female) of varying sizes were examined grossly. Sections (5 µm) from throughout the dorsal and ventral dental pads were stained with Hematoxylin and Eosin to document microanatomy. The thickness of the epithelium and stratum corneum were measured. RESULTS: The ventral dental pad epidermal (1129-3391 µm) and stratum corneum (331-1848 µm) thickness increased with increased body size. The dorsal dental pad epidermal (690-1988 µm) and stratum corneum (121-974 µm) thickness varied relative to size. The dental pad anatomy, including the thickened stratum corneum, indicates an importance similar to molars in grinding and physically breaking up plant material. Extensive appendages including filiform-like papillae and well-developed rete were observed and likely provide physical support for mastication. CONCLUSION: While the sample size limits specific conclusions based on sex or age, it provides a good overview of the anatomy of the dental pads. The manatee is the only mammal known to have a ventral dental pad and the well-developed grinding surfaces demonstrates a crucial role in mastication for these structures. These dental pads should be evaluated during health checks and necropsies and considered in future research on manatee's feeding mechanisms.

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.

Histological characterization of reproductive tract and fetal annexes of the West Indian Manatee (Trichechus manatus) from Brazil / Caracterização histológica do trato reprodutor e anexos fetais do peixe-boi marinho (Trichechus manatus) do Brasil

The West Indian manatee (Trichechus manatus) is one of the most threatened aquatic mammals in Brazil, and is currently classified as "endangered" (MMA). The objective of this study was to characterize histologically the reproductive tract and fetal annexes of stranded manatees in northeastern Brazil. Tissue samples were collected from the reproductive tract of 23 manatees, which were fixed in 10% buffered formalin, processed using standard histological protocols and stained with hematoxylin eosin. We qualitatively described the histological and histomorphometric characteristics of each structure. Six ovaries were analyzed. In four ovaries, we found a large number of primordial and primary follicles. Two ovaries were different from the others: one had inflammatory infiltration and the other had a thickening in the cortex and absence of follicles. We also analyzed seven uteri (of which four were in the proliferative phase, two in the secretory phase, and one in the recovery phase), four placentas, one vagina, six testes (four were in the immature phase, one in the pubertal phase, and one in the mature phase), two epididymides, two penises, and one umbilical cord. The histological and morphometric findings in our work will support future analyses of the reproductive tract of T. manatus from Brazil.(AU)

O peixe-boi marinho (Trichechus manatus) é um dos mamíferos aquáticos mais ameaçados do Brasil e, atualmente é classificado como "em perigo" (MMA). O objetivo deste estudo foi caracterizar histologicamente o trato reprodutor e os anexos fetais de peixes-bois marinhos encalhados no Nordeste do Brasil. Foram coletadas amostras de tecidos do trato reprodutor de 23 peixes-bois marinhos (T. manatus), que foram fixadas em formol tamponado a 10%, processados usando protocolos histológicos padrão e corados com hematoxilina eosina. Foi realizada a descrição qualitativa das características histológicas e histomorfométricas de cada estrutura. Foram analisados seis ovários. Em quatro ovários, foi encontrado um grande número de folículos primordiais e primários. Dois ovários eram diferentes dos outros: um tinha infiltração inflamatória e o outro tinha um espessamento no córtex e ausência de folículos. Também foram analisadas sete amostras uterinas (das quais quatro estavam na fase proliferativa, duas na fase secretória e uma na fase de recuperação), quatro placentas, uma vagina, seis testículos (quatro na fase imatura, um na fase puberal e um na fase madura), dois epidídimos, dois pênis e um cordão umbilical. Os achados histológicos e morfométricos em nosso trabalho apoiarão futuras análises do trato reprodutivo de T. manatus do Brasil.(AU)

Ocorrência de infecção Cryptosporidium spp. em peixe-boi marinho (Trichechus manatus) / Occurrence of Cryptosporidium spp. infection in antillean manatee (Trichechus manatus)

A criptosporidiose constitui-se como uma zoonose que pode afetar o homem e uma ampla variedade de animais domésticos e silvestres, principalmente indivíduos imunodeficientes. O objetivo desse trabalho foi registrar a ocorrência de infecção por Cryptosporidium em peixe-boi marinho. Após ser constatada a mudança de comportamento de um peixe-boi marinho mantido nos oceanários do Centro Mamíferos Aquáticos, ICMBio - FMA, animal foi submetido à exame clínico e, posteriormente, à coleta de amostra fecal. As amostras fecais foram analisadas pela técnica de Kinyoun, teste de imunofluorescência direta e pelo corante 4'.6'-Diamidino-2-Phenilindole (DAPI). No exame clínico, o animal apresentou sinais de desconforto abdominal. Os resultados obtidos nas análises de microscopia de luz e fluorescente revelaram a presença de oocistos de Cryptosporidium nas fezes desse peixe-boi.

Cryptosporidiosis is a zoonosis which can affect man and a wide range of domestic and wild animals, mainly immunodeficient individuals. The objective of this paper was reported the occurrence of a Cryptosporidium infection in Antillean manatee. After an unusual behavior of an Antillean manatee kept in captivity at the Centro Mamíferos Aquáticos, ICMBio - FMA, clinical examination and posterior fecal sampling was performed. Fecal samples were examined by the Kinyoun technique, Direct Immunofluorescence Test and also examined by 4'.6'-Diamidino-2-Phenylindole (DAPI) staining. At the clinical examination, the animal showed signs of abdominal pain. The results obtained by light and fluorescence microscopy analysis showed the presence of Cryptosporidium spp. oocyst in feces of this manatee.