Polymorphic microsatellite DNA markers for the Florida manatee (Trichechus manatus latirostris)

Florida manatees (Trichechus manatus latirostris) are marine mammals that inhabit the coastal waters and rivers of the southeastern USA, primarily Florida. Previous studies have shown that Florida manatees have low mitochondrial DNA variability, suggesting that nuclear DNA loci are necessary for discriminatory analyses. Here we report 10 polymorphic microsatellite loci with an average of 4.2 alleles per locus, and average heterozygosity of 50.1%. These loci have been developed for use in population studies, parentage assignment, and individual identification.     

Evaluation of adrenocortical function in Florida manatees (Trichechus manatus latirostris)

The study objectives were to determine the predominant manatee glucocorticoid; validate assays to measure this glucocorticoid and adrenocorticotropic hormone (ACTH); determine diagnostic thresholds to distinguish physiological vs. pathological concentrations; identify differences associated with sex, age class, female reproductive status, capture time, and lactate; and determine the best methods for manatee biologists and clinicians to diagnose stress. Cortisol is the predominant manatee glucocorticoid. IMMULITE 1000 assays for cortisol and ACTH were validated. Precision yielded intra‐ and inter‐assay coefficients of variation for serum cortisol: ≤23.5 and ≤16.7%; and ACTH: ≤6.9 and ≤8.5%. Accuracy resulted in a mean adjusted R²≥0.87 for serum cortisol and ≥0.96 for ACTH. Assay analytical sensitivities for cortisol (0.1 µg/dl) and ACTH (10.0 pg/ml) were verified. Methods were highly correlated with another IMMULITE 1000 for serum cortisol (r=0.97) and ACTH (r=0.98). There was no significant variation in cortisol or ACTH with sex or age class and no correlation with female progesterone concentrations. Cortisol concentrations were highest in unhealthy manatees, chronically stressed by disease or injury. ACTH was greatest in healthy free‐ranging or short‐term rehabilitating individuals, peracutely stressed by capture and handling. Cortisol concentrations ≥1.0 µg/dl were diagnostic of chronic stress; ACTH concentrations ≥87.5 pg/ml were diagnostic of peracute stress. In healthy long‐term captive manatees, cortisol (0.4±0.2 µg/dl) and ACTH (47.7±15.9 pg/ml) concentrations were lower than healthy free‐ranging, short‐term rehabilitated or unhealthy manatees. Capture time was not significantly correlated with cortisol; ACTH correlation was borderline significant. Cortisol and ACTH were positively correlated with lactate. Zoo Biol 30:17–31, 2011. © 2010 Wiley‐Liss, Inc.     

Detection of hydrodynamic stimuli by the Florida manatee (Trichechus manatus latirostris)

Florida manatees inhabit the coastal and inland waters of the peninsular state. They have little difficulty navigating the turbid waterways, which often contain obstacles that they must circumnavigate. Anatomical and behavioral research suggests that the vibrissae and associated follicle–sinus complexes that manatees possess over their entire body form a sensory array system for detecting hydrodynamic stimuli analogous to the lateral line system of fish. This is consistent with data highlighting that manatees are tactile specialists, evidenced by their specialized facial morphology and use of their vibrissae during feeding and active investigation/manipulation of objects. Two Florida manatees were tested in a go/no-go procedure using a staircase method to assess their ability to detect low-frequency water movement. Hydrodynamic vibrations were created by a sinusoidally oscillating sphere that generated a dipole field at frequencies from 5 to 150 Hz, which are below the apparent functional hearing limit of the manatee. The manatees detected particle displacement of less than 1?μm for frequencies of 15–150 Hz and of less than a nanometer at 150 Hz. Restricting the facial vibrissae with various size mesh openings indicated that the specialized sensory hairs played an important role in the manatee’s exquisite tactile sensitivity.     

Functional morphology of the taste buds of Florida manatee,Trichechus manatus latirostris

The Florida manatee, Trichechus manatus latirostris, is a fully aquatic, threatened marine mammal for which increased understanding of their physiology, reproduction, and nutrition supports management decisions. Manatees may use taste to distinguish saltwater gradients, toxin detection, food assessment, and social interactions. This study sought to locate and characterize manatee taste buds comparing location, structure, number, and size to other species. Entire heads from manatees (6 males, 4 females) of various ages were obtained. The muzzle, tissue surrounding the nares, oral cavity, and epiglottis were examined grossly for pits and papillae. Tissues were examined using light and transmission electron microscopy. Within the predominant taste bud location, the tongue root, taste bud number was estimated using samples from four animals. The average number of taste buds within the tongue root was 11,534 (range 2,711–23,237) with sparse taste buds located on the soft palate and epiglottis. The location along the lateral surface of the tongue root and bordered by grooves, through which tastants could be easily transported, has functional significance. Large numbers of taste buds within the tongue root suggest that taste is an important component of manatee sensory systems and behavioral research would clarify this.     

Characterization of kidney epithelial cells from the Florida manatee, Trichechus manatus latirostris

Summary The West-Indian manatee,Trichechus manatus latirostris, is a herbivorous marine mammal found in the coastal waters of Florida. Because of their endangered status, animal experimentation is not allowed. Therefore, a cell line was developed and characterized from tissue collected during necropsies of the manatees. A primary cell culture was established by isolating single cells from kidney tissue using both enzymatic and mechanical techniques. Primary manatee kidney (MK) cells were subcultured for characterization. These cells were morphologically similar to the cell lines of epithelial origin. An immunocytochemistry assay was used to localize the cytokeratin filaments common to cells of epithelial origin. At second passage, epithelial-like cells had an average population-doubling time of 48 h, had an optimum seeding density of 5×10³ cells/cm², and readily attached to plastic culture plates with a high level of seeding efficiency. Although the epithelial-like cells had a rapid growth rate during the first three passages, the cloning potential was low. These cells did not form colonies in agar medium, were serum dependent, had a limited life span of approximately nine passages, and possessed cell-contact inhibition. These data suggest that the cells were finite (noncontinuous growth), did not possess transformed properties, and were of epithelial origin. These cells are now referred to as MK epithelial cells.     

Circadian and ultradian activity rhythms in manatee (Trichechus manatus manatus) in captivity

In this study, we show temporal organization of activity patterns in larger temporal series recording. The objective of this study was to determine the temporal pattern of the rest-activity rhythm in manatee (Trichechus manatus manatus) in captivity. Activity recordings were programmed from August 2010 to September 2011 with actimetry devices, and behavior recordings were conducted in dry and rainy seasons. We showed that the marine manatee presents a complex temporal organization, in which the rest-activity rhythm comprises several frequencies with a predominant circadian component and multiple ultradian components. Our results indicate that the animals were more active during the day with respect to the night. The temporal organization of this cycle entails multiple frequencies that include ultradian rhythms, which may be expressions generated by physiological needs, such as food availability and thermoregulatory requirements. These patterns should be taken into consideration for future studies of biological rhythms in manatee.     

Composition of the milks of the bottlenose dolphin (Tursiops trucatus) and the Florida manatee (Trichechus manatus latirostris)

Milk samples from four individual bottlenose (Tursiops truncatus) and two Florida manatees (Trichechus manatus latirostris) of known lactation stages were analyzed for protein, carbohydrate and lipid composition, as well as for activity levels of alpha-lactalbumin, the regulatory protein of lactose synthase. The milk from both species had relatively high protein and lipid levels, as reported previously for other marine mammals. The major proportion of the lipid was in the form of triglycerides. Dolphin milk contained an average of 2.2% neutral sugars, which was essentially all in the form of lactose, as determined by several criteria. Manatee milk samples contained 0.6% of neutral sugars, and a larger proportion (about 2%) of amino sugars. Lactose was not detected by enzymatic assay or paper chromatography, but HPLC analysis indicated the presence of low levels of lactose together with two components that were tentatively identified as oligosaccharides. alpha-Lactalbumin activity, determined by assay with bovine galactosyltransferase, was found in both dolphin and manatee milk. The level in dolphin milk was comparable with that found in bovine and other milk, but the level in the manatee was less than 10% of that in the dolphin.     

Tactile discrimination of textures by Florida manatees (Trichechus manatus latirostris)

Two male Florida manatees (Trichechus manatus latirostris) demonstrated sensitive tactile discrimination in a two‐alternative forced choice task, using a modified staircase method. Stimuli were acrylic plates with vertical gratings of ridges and grooves. The standard stimulus, present on every trial, had 2 mm gratings and the comparison stimuli had wider gratings. The blindfolded subjects were trained to demonstrate discrimination by pressing the target with wider gratings. Discrimination thresholds (75% correct) for the subjects were 2.05 mm and 2.15 mm, corresponding to Weber fractions of 0.025 and 0.075, respectively. These results indicate thresholds on similar stimuli comparable to humans (index finger tasks) and better than harbor seals, Phoca vitulina, and the closely related Antillean manatee, Trichechus manatus manatus. Memory for the tactile task was quite stable for both subjects, over 2 yr in the case of one of the subjects. Video analysis of responses indicated that bristle‐like hairs, perioral bristles, and skin on the oral disk were involved in the discrimination response.     

Phylogeography of the West Indian manatee (Trichechus manatus): how many populations and how many taxa?

To resolve the population genetic structure and phylogeography of the West Indian manatee ( Trichechus manatus ), mitochondrial (mt) DNA control region sequences were compared among eight locations across the western Atlantic region. Fifteen haplotypes were identified among 86 individuals from Florida, Puerto Rico, the Dominican Republic, Mexico, Colombia, Venezuela, Guyana and Brazil. Despite the manatee's ability to move thousands of kilometres along continental margins, strong population separations between most locations were demonstrated with significant haplotype frequency shifts. These findings are consistent with tagging studies which indicate that stretches of open water and unsuitable coastal habitats constitute substantial barriers to gene flow and colonization. Low levels of genetic diversity within Florida and Brazilian samples might be explained by recent colonization into high latitudes or bottleneck effects. Three distinctive mtDNA lineages were observed in an intraspecific phylogeny of T. manatus , corresponding approximately to: (i) Florida and the West Indies; (ii) the Gulf of Mexico to the Caribbean rivers of South America; and (iii) the northeast Atlantic coast of South America. These lineages, which are not concordant with previous subspecies designations, are separated by sequence divergence estimates of d = 0.04–0.07, approximately the same level of divergence observed between T. manatus and the Amazonian manatee ( T. inunguis , n = 16). Three individuals from Guyana, identified as T. manatus , had mtDNA haplotypes which are affiliated with the endemic Amazon form T. inunguis . The three primary T. manatus lineages and the T. inunguis lineage may represent relatively deep phylogeographic partitions which have been bridged recently due to changes in habitat availability (after the Wisconsin glacial period, 10 000 BP ), natural colonization, and human-mediated transplantation.     

PERMANENT GENETIC RESOURCES: Eighteen new polymorphic microsatellite markers for the endangered Florida manatee, Trichechus manatus latirostris

Here we describe 18 polymorphic microsatellite loci for Trichechus manatus latirostris (Florida manatee), isolated using a polymerase chain reaction‐based technique. The number of alleles at each locus ranged from two to four (mean = 2.5) in specimens from southwest (n = 58) and northeast (n = 58) Florida. Expected and observed heterozygosities ranged from 0.11 to 0.67 (mean = 0.35) and from 0.02 to 0.78 (mean = 0.34), respectively. Departures from Hardy–Weinberg equilibrium occurred at two loci. There was no evidence of genotypic disequilibrium for any pair of loci. For individual identification, mean random‐mating and θ‐corrected match probabilities were 9.36 × 10^?7 and 1.95 × 10^?6, respectively.     

Helminths of the Florida manatee, Trichechus manatus latirostris, with a discussion and summary of the parasites of sirenians

We examined 215 Florida manatees (Trichechus manatus latirostris) at necropsy to determine the helminth fauna. Six species were identified: Heterocheilus tunicatus (Nematoda: Ascaridoidea); Anoplocephala sp. (Cestoda: Cyclophyllidea); and 4 species of trematodes, Cochleotrema cochleotrema (Digenea: Opisthotrematidae), Chiorchis fabaceus (Digenea: Paramphistomatidae), Nudacotyle undicola (Digenea: Nudacotylidae), and Moniligerum blairi (Digenea: Opisthotrematidae). Seventy-three percent of the manatees examined were infected with at least 1 species of helminth. The mean number of species of helminths per infected manatee was 1.9 with a range of 1-4. Fifty-nine manatees were helminth-free; 30 of these were calves. No associations were found between the intensity of helminth infections and host sex, age class, season, and geographic location of recovery, or cause of death. Differences in parasite prevalence between age classes were highly significant for Chiorchis, Cochleotrema, and Heterocheilus, due to a low number of infected calves. A higher prevalence of Cochleotrema was found in manatees recovered from eastern Florida, and Heterocheilus was evident in significantly more manatees from western and souther Florida. Comparisons in the parasite fauna are made among Florida manatees and other sirenian populations, and a brief review of sirenian parasites is included.     

Seroepidemiology of TmPV1 infection in captive and wild Florida manatees (Trichechus manatus latirostris)

In 1997, cutaneous papillomatosis caused by Florida manatee (Trichechus manatus latirostris [Tm]) papillomavirus 1 (TmPV1) was detected in seven captive manatees at the Homosassa Springs Wildlife State Park, Florida, USA, and, subsequently, in two wild manatees from the adjacent Homosassa River. Since then, papillomatosis has been reported in captive manatees housed in other locations, but not in wild animals. To determine TmPV1 antibody prevalence in captive and wild manatees sampled at various locations throughout Florida coastal regions, virus-like particles, composed of the L1 capsid protein of TmPV1, were generated with a baculovirus expression system and used to measure anti-TmPV1 antibodies in an enzyme-linked immunosorbent assay. Serologic analysis of 156 manatees revealed a TmPV1 antibody prevalence of 26.3%, with no significant difference between captive (n=39) and wild (n=117) manatees (28.2% and 25.6%, respectively). No antibody-positive wild animal showed PV-induced cutaneous lesions, whereas papillomatosis was observed in 72.7% of antibody-positive captive manatees. Our data indicate that Florida manatees living in the wild are naturally infected by TmPV1 but rarely show TmPV1-induced papillomatosis. Hence, it appears that the wild population would not be harmed in a case of contact with captive animals without visible lesions and productive infections, which could be thus released into the wild.     

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.     

Malignant lymphoma in a West Indian manatee (Trichechus manatus)

We identified a malignant lymphoma infiltrating the lung, liver, kidney, mesenteric lymph nodes, and eye as the cause of death in a male West Indian manatee (Trichechus manatus). Diagnosis was based on gross, histopathologic, and immunohistochemical studies. Tissue samples from ten organs were included in a tissue microarray and sections from this array were subjected to immunohistochemical staining. The cytoplasm of the neoplastic lymphocytes identified in six organs was positive for CD3, a marker for T-cell differentiation. The neoplastic cells were negative for CD79alpha, a marker for B-cell differentiation. The cause of this neoplasm was not determined. This is the first report of malignant lymphoma in the mammal order Sirenia.     

Dietary exposure to harmful algal bloom (HAB) toxins in the endangered manatee (Trichechus manatus latirostris) and green sea turtle (Chelonia mydas) in Florida,USA

Florida is a hotspot for cyano- and microalgal harmful algal blooms (HABs) with annual red-tide events off-shore and blooms of Lyngbya spp. commonly observed in both marine and freshwater environments. This region also provides extensive foraging habitat for large populations of herbivorous green turtles (Chelonia mydas) and manatees (Trichechus manatus latirostris). The exposure of aquatic organisms to HAB toxins is not well known and whilst acute exposures are better understood, the vulnerability of aquatic animals to chronic exposure from multiple HAB toxins over prolonged periods has rarely been addressed. This study aimed to identify the presence of toxic compounds produced by HAB species commonly found in Florida (brevetoxins, okadaic acid, saxitoxins and Lyngbya toxins) in tissues and gut samples from manatee and green sea turtles that stranded in Florida, USA. Muscle, liver and alimentary tract samples were opportunistically collected from 14 manatees and 13 green turtles that stranded on the Florida shoreline between December 2003 and February 2006. Samples from each animal were assessed for the presence of brevetoxin, okadaic acid, lyngbyatoxin-A and saxitoxin. Nine (64%) manatees and 11 (85%) turtles were found to have been exposed to one or more of the HAB toxins. Okadaic acid and saxitoxin were only found in alimentary tract samples, whereas brevetoxin was more widely distributed. No lyngbyatoxin-A was observed in any tissue samples. The majority of turtles (13) stranded on the Atlantic coast between St. Johns and Monroe counties, with one on the Gulf coast at Bay County, whereas nine manatees were stranded on the Gulf coast between Levy and Lee counties, with the remaining five between Volusia and Brevard counties on the Atlantic coast. This HAB toxin screen has identified that a large proportion of a random sample of turtles and manatees that stranded in Florida in 2003–2006 were exposed to HAB toxins. Most of the concentrations measured were low, and the toxins were directly linked to the death of only three of these animals. However, the presence of these compounds, and in some cases the presence of multiple HAB toxins in individual animals, indicates that turtles and manatees in Florida are exposed to deleterious compounds at sub-lethal levels in their environment, which could ultimately compromise their health.     

Digesta passage and fiber digestibility in captive white-faced sakis (Pithecia pithecia)

Wild white-faced sakis (Pithecia pithecia) ingest primarily seeds that provide a diet that is lipid-rich and moderately high in dietary fiber. Although little anatomical information is available on sakis, evidence from other vertebrate seed predators suggests that such a diet is correlated with adaptations in gut morphology or physiology. Milton [1984] reported a 20 hr transit time (TT=transit time or time of first appearance in feces) of a particulate marker for a single monk saki (Pithecia monachus). This suggests that TT for Pithecia sakis may be four to five times longer than what has been reported for soft-fruit-eating platyrrhines, such as Ateles and Cebus. During a captive study, we calculated an average TT of 14.7+/-0.4 hr (n=5 trials started in the evening) for a chromic oxide (Cr(2)O(3)) marker that follows liquid digesta and TTs of 14.5 hr (trial started the previous evening) and 23.0 hr (trial started the previous morning) for two trials using particulate markers. Mean retention time (MRT) for the liquid marker ranged from 15.3 hr to 37.7 hr in four trials that lasted longer than 90 hr. Marker recovery was incomplete for the particulate markers in these trials, and thus MRT could only be determined for the liquid phase marker. Three 5-day trials on a low-fiber, blended diet revealed high fiber fraction digestibilities (neutral detergent fiber (NDF)=77.4% and acid detergent fiber (ADF)=74.4%). Data collected for this study and nutritional data from wild sakis suggest that pitheciin seed predators may have a potential for fiber digestibility that is intermediate between ripe-pulp frugivores and folivores.